Cancer-associated genes

ABSTRACT

To provide a method for detecting a cancer cell in a resected specimen by determining a change in an expression level of at least one of cancer-associated genes selected from genes of which cDNA is a DNA comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, or a DNA capable of hybridizing with a nucleic acid consisting of a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing under stringent conditions; as well as a kit for detecting cancer by the above method, and the like.

[0001] This application is a continuation-in-part application ofPCT/JP98/00667, the entire contents of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for detecting a cancercell characterized by detecting an expression product of a gene capableof changing an expression level thereof owing to canceration. Thepresent invention relates to a gene capable of changing an expressionlevel thereof and a product of the gene owing to canceration.

[0004] 2. Discussion of the Related Art

[0005] Cancers constitute the top of the causes for mortality in Japansince 1981, and a gastric cancer occurs especially at the highestfrequency. Recently, there has been known that there is a multi-stagecarcinogenic mechanism in the course from a normal cell to a cancer cell[Fearon, E. R. et al., Cell, 61, 759-767 (1990); Sugimura, T., Science,258, 603-607 (1992)] for which the accumulation of the abnormality in aplurality of genes including DNA repair genes, tumor suppressor genesand oncogenes is essential. Generally, the instability of a gene and theinactivation of a tumor suppressor gene are involved in the developmentof a cancer, and the activation of an oncogene and/or the overexpressionof a growth factor are involved in the advancement and malignancy of acancer.

[0006] The instability of a gene includes the instability of geneassociated with abnormality in a DNA mismatch repair system and theinstability at a chromosomal level. An example of the former includesthe difference in the chain length of a simple repeated sequence presentin a genome between a cancer site and a non-cancer site in the sameindividual (microsatellite instability) [Thibodeau, S. N. et al.,Science, 260, 816-819 (1993)], and an example of the latter includes aninterchromosomal translocation. The interchromosomal translocation maycause to express a protein which is not found in normal cells, or theinterchromosomal translocation may affect an expression level of aprotein, even if it is expressed in normal cells. In fact, in humanchronic myelocytic leukemia, bcr gene is fused with c-abl gene by theinterchromosomal translocation, and there has been confirmed anexpression of a hybrid mRNA transcribed from bcr-abl fusion gene, whichis absent in normal cells. Further, there has been confirmed that anintroduction of bcr-abl fusion gene into an animal results in an onsetof leukemia [Watson, J. D. et al., Molecular Biology of RecombinantDNAS, 2nd Ed., Maruzen K. K., 309 (1992)].

[0007] The inactivation of a tumor suppressor gene includes, forexample, an inactivation of p53 gene. The inactivation is considered tobe caused by a deletion within the gene, or a point mutation occurringin a particular portion of an encoding region [Nigro, J. M. et al.,Nature, 342, 705-708 (1989); Malkin, D. et al., Science, 250, 1233-1238(1990)]. In addition, since the deletion and the point mutation of thep53 gene are observed in various cancers, and are as frequent as 60% orhigher especially in cases of a gastric cancer at an early stage[Yokozaki, H. et al., Journal of Cancer Research and Clinical Oncology,119, 67-70 (1992)], the detection of these mutations is considered to beuseful for detecting a cancer at an early stage.

[0008] On the other hand, p16/MTS1 gene has been known to be a genewhich is inactivated owing to a homologous deletion, and high-frequencyhomologous deletions have been observed in cases of a glioma, apancreatic cancer and a urinary bladder cancer [Cairns, P. et al.,Nature Genetics, 11, 210-212 (1995)]. p16 Protein regulates a cellcycle, and the abnormality in p16 expression has been suggested to beinvolved in the canceration of a cell [Okamoto, A. et al., Proceedingsof the National Academy of Sciences of the United States of America, 91,11045-11049 (1994)].

[0009] As the causation for the activation of an oncogene, there can beincluded, for example, a viral insertion mutation in a proximity of anoncogene and an interchromosomal translocation. For example, a viralinsertion mutation has been confirmed in lymphoma of a chicken which iscaused by an avian leukosis virus (ALV). In this case, it has been foundthat DNA of an ALV is inserted in the proximity of a gene c-myc, and, bypotent viral enhancer and promoter, a normal c-myc is overexpressed, anda new sequence which is different partially from the normal gene hasbeen expressed. In addition, in a certain kind of human B cell tumor,there has been confirmed that c-myc, which is one of oncogenes, islocated near a potent transcription signal of immunoglobulin by theinterchromosomal translocation, whereby increasing its expression levelof the mRNA. In this case, no difference has been found between aprotein for c-myc in a cancer cell and a protein for c-myc expressed ina normal cell, and the canceration is considered to be caused by anincrease in the expression level of the c-myc mRNA [Watson, J. D. etal., Molecular Biology of Recombinant DNAS, 2nd Ed., Maruzen K. K.,305-308 (1992)].

[0010] An overexpression of a growth factor includes, for example, anoverexpression of C-Met which encodes a hepatocyte growth factorreceptor. There has been confirmed that the abnormality in expression ofthe C-Met is observed as an expression of mRNA having the length of 6.0kb which is not found in a normal mucous membrane at an early stage ofgastric cancer [Kuniyasu, H. et al., International Journal of Cancer,55, 72-75 (1993)], or is observed at a high frequency, and that acorrelation between the gene amplification and the degree of the cancermalignancy is observed [Kuniyasu, H. et al., Biochemical and BiophysicalResearch Communications, 189, 227-232 (1992)].

[0011] As examples of confirming the correlation between the geneabnormality and the degree of cancer malignancy, in addition to that ofthe c-Met mentioned above, there have been confirmed that anamplification and/or an overexpression of an oncogene C-erbB2 gene isfound in mammary cancers, ovarian cancers, gastric cancers and uterinecancers [Wright, C. et al., Cancer Research, 49, 2087-2090 (1989);Saffari, B. et al., Cancer Research, 55, 5693-5698 (1995)]; and that anamplification and/or an overexpression of an oncogene K-sam gene isfound in a poorly-differentiated adenocarcinoma which is one tissue typeof gastric cancer [Tahara, E. et al., Gastric Cancer, Tokyo,Springer-Verlag, Published in 1993, 209-217], respectively.

[0012] As described above, the information concerning the gene involvedin the development and the advancement of a cancer as well as theabnormality of such genes has been increasing, and the genetic diagnosisof a biopsy material may serve for an early diagnosis and an assessmentof the degree of malignancy of a cancer. However, since a carcinogenicmechanism comprises multiple steps and requires an accumulation of aplurality of mutations, a large part of the genes associated with thecanceration have still yet been unknown, and further study is necessary.Recently, a gene therapy in which a normal p53 gene is introduced into acancer cell whereby suppressing the proliferation of the cancer cell isnow at a stage of a clinical trial. Therefore, the solution for acancer-suppressing gene can shed light not only in the diagnosis butalso in the gene therapy.

SUMMARY OF THE INVENTION

[0013] Accordingly, a first object of the present invention is toprovide a method for detecting cancerated cell and a method fordetermining a degree of malignancy, on the basis of finding a geneusable as an index for carcinogenesis, particularly a gene capable ofchanging expression conditions thereof by canceration of a cell, andmeasuring an expression level of the gene in a resected specimen. Asecond object of the present invention is to provide a kit used for theabove method for detecting a cancer cell and/or a method for determininga degree of malignancy of the cell. A third object of the presentinvention is to provide a method for controlling proliferation of acancer cell by using a substance specifically binding to a gene capableof serving as an index for carcinogenesis or an expression product ofthe gene. Furthermore, a fourth object of the present invention is toprovide a novel peptide associated with canceration, and a nucleic acidencoding the peptide. These and other objects of the present inventionwill be apparent from the following description.

[0014] To summarize the present invention, a first invention of thepresent invention is an invention pertaining to a method for detecting acancer cell in a resected specimen, characterized by determining achange in an expression level of a gene selected from genes of whichcDNA corresponds to a DNA comprising a nucleotide sequence as shown inany one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, or aDNA capable of hybridizing with a nucleic acid as shown in any one ofSEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing under stringentconditions by, for example, determining a change of an expression levelof mRNA or a change of a protein expression level.

[0015] A second invention of the present invention is an inventionpertaining to a kit for detecting cancer by the method for detecting ofthe present invention, characterized in that the kit comprises as anessential constituent any one of primers for amplifying mRNA as an indexfor a change in an expression level, a probe capable of hybridizing withthe above mRNA, or an antibody recognizing a protein as an index for thechange in expression level.

[0016] A third invention of the present invention is a method forcontrolling proliferation of a cancer cell by using a substancespecifically binding to the gene or an expression product thereof,characterized in that cDNA of the gene corresponds to a DNA comprising anucleotide sequence any one of sequences of SEQ ID NOs: 1 to 16 and 66to 68 in Sequence Listing, or a DNA capable of hybridizing with DNA asshown in any one of sequences of SEQ ID NOs: 1 to 16 and 66 to 68 inSequence Listing, wherein the method gives transcriptional control ofthe gene and/or functional control of an expression product thereof, andthe like.

[0017] A fourth invention of the present invention is an inventionpertaining to a peptide usable for detecting cancer and a nucleic acidencoding the peptide, characterized in that the peptide consists of anamino acid sequence comprising an entire portion of an amino acidsequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and 70 inSequence Listing or a partial portion thereof and a nucleic acidencoding the peptide.

[0018] A fifth invention of the present invention pertains to anantibody usable for detecting cancer, the antibody recognizing the abovepeptide of the fourth invention.

[0019] Incidentally, the term “resected specimen” used in the presentspecification refers to blood, urine, feces, tissue resected by surgery,and the like. On the other hand, the term “cancer-associated gene”refers to a gene in which the expression conditions thereof change withcanceration of a cell.

[0020] In order to achieve the objects mentioned above, the presentinventors have found a cancer-associated gene by comparing theintracellular expression levels of genes between a cancer tissue and acontrol normal tissue of a cancer patient, and they have found thatcancer cells can be detected by comparing the expression level of thisgene. In addition, they have found a novel gene in thiscancer-associated gene, whereby completing the present invention.

[0021] The terms “cancer tissue” and “control normal tissue” used in thepresent specification mean a tissue constituting a region of cancerouslesion in a multicellular individual and a tissue constituting a regionwhich is identical spatially to the cancer tissue in the same individualbut functions normally.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is an autoradiogram showing electrophoretic patterns of theresulting DNA fragment in a case of detecting cancer-associated genes byDD method.

[0023]FIG. 2 is an autoradiogram obtained by electrophoresing RNA andthen hybridizing a labeled probe with a desired mRNA, in a case ofdetecting a change in an expression level of mRNA of cancer-associatedgenes by Northern hybridization method.

[0024]FIG. 3 is a picture showing electrophoretic patterns of theresulting DNA fragment in a case of detecting a change of expression ofa cancer-associated gene by RT-PCR method.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be explained concretely below.

[0026] The first invention of the present invention provides a methodfor detecting a cancer cell using an expression level of thecancer-associated gene as an index.

[0027] A gene which can serve as an index for canceration is a genecapable of changing expression conditions thereof by canceration of acell, namely, a gene of which expression is significantly induced orsuppressed. Such a gene can be detected by, for instance, analyzing copynumber of the gene on genome or a pattern for translocation inchromosomes, and comparing an expression level of a gene product in anormal cell and a cancerated cell to identify a gene having differencesin both cells. The gene product includes, for example, mRNA transcribedby the above gene or a protein which is a translational product. In thedetection in the present invention for a cancer-associated gene, it isefficient to use as an index an expression level of mRNA, in whichvarious methods have been developed for its analysis with the progressin gene manipulation technique. Procedures for confirming a change in anexpression level of a gene using as an index an expression level of mRNAincludes subtractive hybridization method [Zimmermann, C. R. et al.,Cell, 21, 709-715 (1989)], Representational Difference Analysis (RDA)method [Lisitsyn, N. et al., Science, 259, 946-951, (1993)], molecularindex method (Japanese Patent Laid-Open No. Hei 8-322598), DifferentialDisplay (DD) method [Liang, P. and Pardee, A. B., Science, 257, 967-971(1992)], and the like. Among them, since the procedures of the DD methodare simple, the DD method is suitable for screening a gene in thepresent invention. The method for screening a cancer-associated gene byusing the DD method utilized in the present invention will be describedin detail below.

[0028] First, mRNA is converted to cDNA by carrying out a reversetranscription reaction with a genome DNA-removed crude RNA sampleresulting from treating each RNA individually extracted from a cancertissue and a control normal tissue to be compared with DNase, togetherwith an oligo(dT) anchor primer and a reverse transcriptase (RTase).Thereafter, the nucleic acid amplification is carried out by polymerasechain reaction (PCR) with the oligo(dT) anchor primer in combinationwith various random primers.

[0029] Subsequently, a PCR-amplified product obtained separately fromthe tissues to be compared is subjected to polyacrylamideelectrophoresis for each amplified product resulting from a combinationof an identical primer pair. The band patterns are compared with eachother to find a band exhibiting a difference between the normal cell andthe cancer cell. This band is cut out from the gel, and a nucleic acidcontained in the band is extracted, whereby a DNA fragment which isconsidered to be complementary to a partial portion with the mRNA forthe cancer-associated gene can be obtained.

[0030] Thereafter, there is studied whether changes in expression levelsof mRNA for the cancer-associated gene can be truly confirmed from theDNA fragment obtained in the DD method described above. When theexpression level of the mRNA in a normal tissue is confirmed to behigher than that in the cancer tissue, it is determined that thecancer-associated gene is a gene of which expression level is reducedowing to canceration. On the other hand, when the expression level ofthe mRNA in the cancer tissue is confirmed to be higher than that in thenormal tissue, it is determined that the cancer-associated gene is agene of which expression level is amplified owing to canceration.

[0031] The confirmation on an expression level of mRNA can be made, forexample, by labeling the DNA fragment obtained, subjecting a crude RNAsample extracted from either of the cancer tissue or the control normaltissue to Northern hybridization using the above DNA fragment as adetection probe, and confirming the difference in the observed signalintensity with a densitometer. In other words, the stronger the signalintensity, it can be determined that the expression level of the mRNA ishigh. For example, a signal intensity can be expressed as a value for avolume of a band [IOD (Integrated Optical Density)] obtained from anautoradiogram, or the like. Here, the higher the IOD value, it can bedetermined that the expression level of the mRNA corresponding to theband is high.

[0032] When the expression level of mRNA is too low so that the changein the expression level of the mRNA cannot be confirmed by means ofNorthern hybridization analysis, there can be also confirmed with moresensitive RNase protection assay [Krieg, P. A. and Melton, D. A.,Methods in Enzymology, 155, 397-415 (1987)] using as a probe RNAprepared from an amplified DNA fragment obtained by the DD methoddescribed above, which is derived from mRNA deduced to be expressed froma cancer-associated gene as a template. This method utilizes RNasehaving substrate specificity wherein it shows cleaving activity onsingle-stranded RNA, but shows no cleaving activity on double-strandedRNA. Specifically, an excessive amount of a probe is added to a crudeRNA sample extracted from a normal tissue and a cancer tissue-derivedcrude RNA sample, and the mRNA to be detected forms a hybrid with theadded probe, whereby acting on an RNase having substrate specificity.The expression level of the mRNA can be confirmed by determining theamount of the double-stranded RNA remaining after the digestion with theRNase mentioned above. In other words, the larger the amount of theremaining double-stranded RNA, it can be determined that the expressionlevel of the mRNA is high.

[0033] The nucleotide sequence of an amplified DNA fragment obtained bythe DD method described above, which is derived from mRNA deduced to beexpressed from a cancer-associated gene as a template, is sequenced byPCR direct sequencing [Erlich, H. A., PCR Technology, Stockton Press,Published in 1989, 45-60], or by a combination of TA cloning [Mead, D.A. et al., Bio/Technology, 9, 657-663 (1991)] with a usual nucleotidesequencing method to determine the nucleotide sequence, and the amountsof the amplified product as obtained by carrying out RT-PCR with anamplification primer which is designed based on the above nucleotidesequence information are then compared, whereby the mRNA expressionlevel can be confirmed. In other words, the higher the amount of theresulting amplified product, it can be determined that the expressionlevel of the mRNA is high.

[0034] Incidentally, the amplified DNA fragment obtained by the DDmethod described above, which is derived from mRNA deduced to beexpressed from a cancer-associated gene as a template, is notnecessarily cDNA complementary to an entire length of mRNA for thecancer-associated gene. In order to obtain cDNA for a cancer-associatedgene, for example, a cDNA library derived from a tissue used inscreening is prepared; an amplified DNA fragment obtained by the DDmethod described above, which is derived from mRNA deduced to beexpressed from a cancer-associated gene as a template, is labeled; andDNA derived from plaque hybridization is carried out with the labeledcancer-associated gene as a detection probe, whereby cDNA clone for acancer-associated gene can be isolated.

[0035] The present inventors have succeeded in isolating 14 kinds of DNAfragments comprising a respective nucleotide sequence of a partialportion of cDNA for cancer-associated genes. Genes expressing mRNA whichcorresponds to cDNA as shown in nucleotide sequences comprising anucleotide for the DNA fragment thus obtained are named as CA11, CA13,CC24, GG24, AG26, GC31, GC32, GC33, GG33, CC34, GC35, GC36, CA42 andCC62, respectively. Correspondences between SEQ ID NOs in SequenceListing in which a nucleotide sequence of regions presently determinedin each nucleotide sequence of cDNA for 14 kinds of cancer-associatedgenes and the above name of the gene named by the present inventors areshown in Table 1. TABLE 1 SEQ ID NOs in Sequence Listing NucleotideAmino Acid Sequence Sequence Name of Gene  1, 66 17, 69 CA11 2 18 CA13 3CC24 4 GG24 5 AG26 6 GC31 7 GC32 8 GC33 9 GG33 10  CC34 11, 67 GC35 12,15, 16, 68 70 GC36 13  19 CA42 14  CC62

[0036] Here, in Table 1, the nucleotide sequence as shown in SEQ ID NO:68 comprises the sequences as shown in SEQ ID NOs: 12, 15 and 16. Inaddition, the amino acid sequence as shown in SEQ ID NO: 70 is a deducedsequence based on the nucleotide sequence as shown in SEQ ID NO: 68.

[0037] The above cancer-associated genes are roughly classified into agene in which the expression level is decreased or increased bycanceration. The former genes include CA11, AG26, GC35, GC36 and CC62;and the latter genes include CA13, CC24, GG24, GC31, GC32, GC33, GG33,CC34 and CA42.

[0038] By comparing the expression level of each of the genes obtainedas above, cancer cells can be detected. In this case, thecancer-associated gene serving as an index may be appropriately selectedfrom the genes listed above, and it may be used as a single kind, or incombination of several kinds of genes. In addition, thecancer-associated gene serving as an index for detection of a cancercell is not particularly restricted to the 14 kinds of genes listedabove, and the cancer-associated gene may be any gene of which cDNA isDNA capable of hybridizing under stringent conditions with the DNA asshown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in SequenceListing, as long as the expression level of the gene is changed owing tocanceration of a cell.

[0039] Conditions capable of hybridizing used in the presentspecification refer to, for example, those capable of hybridizing by aprocess comprising incubating DNA immobilized on a nylon membrane with aprobe at 65° C. for 20 hours in a solution containing 6×SSC (wherein1×SSC is a solution prepared by dissolving sodium chloride 8.76 g andsodium citrate 4.41 g in IL of water), 1% SDS, 100 μg/ml herring spermDNA, 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone and 0.1%Ficol.

[0040] In fact, there has also been confirmed in the present inventionthe presence of a gene having the characteristics described above. Thenucleotide sequence as shown in SEQ ID NO: 10 in Sequence Listing ispresent in the nucleotide sequence of cDNA for CC34 gene. DNA as shownin this nucleotide sequence wherein T at base number 935 of the sequenceis substituted with A, and 6 bases consisting of the sequence of GTTAAGat a 3′-terminal are deleted has been obtained as a DNA fragment withdifferent amplification levels in the DD method using RNA prepared froma normal tissue and RNA prepared from a cancer tissue. This amplifiedDNA fragment is capable of hybridizing with DNA as shown in SEQ ID NO:10 in Sequence Listing. Therefore, a gene expressing mRNA which yieldsthis DNA fragment obtained by the DD method in the present invention isalso encompassed in the cancer-associated gene for detecting a cancercell in the present invention.

[0041] In addition, as a result of Northern hybridization using highlypurified mRNA, it is found that there are plural gene transcriptionalproducts capable of hybridizing with GC36 under stringent conditions,and signals corresponding to each of about 2 kb band, and about 2.4 toabout 2.6 kb band are detected in a gastric tissue. In a case of GC35,as a result of Northern hybridization in the same manner as GC36, it isshown that there are plural gene transcriptional products capable ofhybridizing with GC35 under stringent conditions and signalscorresponding to each of about 1.6 kb; about 3.6 to about 4.0 kb; about4.5 kb; and about 5.6 to about 6.0 kb are detected in a gastric tissue.It is considered that these mRNAs result from alternative splicing,wherein mRNAs with different sizes are produced by splicing viadifferent combinations of plural exons of primary transcript (mRNAprecursor) from the same gene. For instance, a nucleotide sequence ofcDNA for nCL-4 encoding digestive tract-specific calpain has highhomology with a nucleotide sequence of cDNA for GC36 gene, wherein thenucleotide sequence of cDNA for nCL-4 was clarified at the date afterthe priority date of the present application [Lee, H. -J. et. al.,Biological Chemistry, 379, 175-183, 1998]. In addition, since GC36 genetranslation product is identical to nCL-4 except for substitution of oneamino acid and deletion of the following 26 amino acids in its aminoacid sequence, it is suggested that the mRNA deduced to be expressedfrom nCL-4 gene and the mRNA deduced to be expressed from GC36 gene areproduced by alternative splicing. Further, in the present invention, itis confirmed that an expression level of the mRNA deduced to beexpressed from nCL-4 gene is reduced by canceration as in the mRNAdeduced to be expressed from GC36 gene. Therefore, the cancer-associatedgene usable for detection of cancer cells in the present invention alsoencompasses mRNAs resulting from alternative splicing, such as the mRNAdeduced to be expressed from nCL-4 gene.

[0042] The determination of whether or not a cell is a cancer cell iscarried out by firstly using a plurality of normal tissues to confirm anormal level of the expression level of the cancer-associated gene usedas an index for canceration by a suitable detection method; subsequentlydetermining an expression level of the cancer-associated gene in aresected specimen; and comparing it with the normal level. Specifically,in a case where the expression of the cancer-associated gene as an indexis suppressed by canceration, it is determined to be cancer-positivewhen the expression of this cancer-associated gene cannot be confirmedor can be confirmed only at a level lower than the normal level in aresected specimen. On the contrary, in a case where the expression ofthe cancer-associated gene as an index is amplified by canceration, itis determined to be cancer-positive when the expression of thiscancer-associated gene is at a level higher than the normal level. Inthe comparison of the expression level of the cancer-associated gene,there may be employed either the amount of mRNA or the amount of aprotein expressed from this gene. Incidentally, the normal levelreferred in the present specification can be shown by the followingequation based on the expression level of the cancer-associated gene ina plurality of normal tissues obtained by an appropriate detectionmethod.

[Normal Level Value]=[Mean Expression Level of Cancer-Associated Gene inNormal Tissue]±2×[Standard Deviation]  Equation 1

[0043] This normal level value as calculated encompasses 95% of thenormal tissues for which the expression level of the cancer-associatedgene is determined.

[0044] The detection method utilizing mRNA includes, for example, RT-PCRmethod, RNase protection assay or Northern hybridization.

[0045] RT-PCR (Reverse transcribed-Polymerase chain reaction) methodrefers to a method comprising synthesizing cDNA by reversetranscriptional reaction using mRNA as a template, and thereafterperforming nucleic acid amplification by PCR [Kawasaki, E. S. et al.,Amplification of RNA. In PCR Protocol, A Guide to Methods AndApplications, Academic Press, Inc., San Diego, 21-27 (1991)]. In thepresent invention, nucleic acid amplification reaction is notparticularly limited, and may be Strand Displacement Amplification (SDA)method [Walker, G. T., Nucleic Acids Res., 20, 1691-1696 (1992)],Nucleic Acid Sequence-Based Amplification (NASBA) method [Compton, J.,Nature, 350, 91-92 (1991)], and the like, in which their reactionconditions are also not particularly limited. In addition, the amplifiedregion of cDNA for the cancer-associated gene is not necessarily anentire length of cDNA, but it may be a partial region of the cDNA, aslong as the confirmation of the amplified products is not hindered. Itis preferable that a primer pair used in nucleic acid amplificationreaction is designed so as to specifically amplify only the cDNA. Aslong as the confirmation of amplified products for the region is nothindered, it does not matter that cDNA which is not subject to detectionmay be amplified. Incidentally, the term “primer” in the presentspecification refers to an oligonucleotide capable of acting as aninitiation site for DNA synthesis in a case of hybridizing with atemplate nucleic acid at a suitable temperature under conditions forallowing initiation of synthesis of a primer extension product by DNApolymerase, namely, in the presence of 4 kinds of different nucleotidetriphosphates and DNA polymerase in suitable buffer (the buffer beingdetermined by pHs, ionic strength, cofactors, and the like). Typically,the primer comprises 10 to 30 nucleotides. For instance, in a case ofCA11 gene in the present specification, there can be exemplified as theformer primer a combination of DNAs as shown in SEQ ID NOs: 20 and 21 inSequence Listing. Hindrance in the confirmation of the amplifiedproducts used in the present specification refers, for instance, to acase where the confirmation is carried out by subjecting the amplifiedDNA fragment to agarose gel electrophoresis, and thereafter staining thegel with ethidium bromide (EtBr), the amount of the amplified DNAfragment present corresponding to mRNA for a cancer-associated gene tobe detected cannot be confirmed, since a large number of the amplifiedDNA fragments having about the same number of bases are produced bynucleic acid amplification reaction, and the separation of eachamplified DNA fragment from each other is incomplete.

[0046] Amounts of the amplified DNA level can be confirmed by subjectingthe nucleic acid amplification reaction mixture to agarose gelelectrophoresis; and confirming from the position and the signalintensity of a band detected with a labeled probe capable ofspecifically hybridizing with a desired amplified fragment. Therefore,the higher the signal intensity obtained by using a certain amount of acrude RNA sample extracted from a resected specimen, it can bedetermined that the expression level of a cancer-associated gene to bedetected is high. The label on the probe is not particularly limited.For example, there can be used a radioactive substance typicallyexemplified by ³²P, or a fluorescent substance typically exemplified byfluorescein. The signal intensity can, for example, be indicated by IODof a band on an autoradiogram or a fluorescent image obtained by themethod described above.

[0047] On the other hand, when an amplified product can be obtained in asufficient amount, the amplified product can be confirmed by subjectingit to agarose gel electrophoresis, staining the gel with EtBr, andconfirming from the position of the amplified DNA fragment and itsfluorescent intensity. Therefore, the higher the fluorescent intensity,it can be determined that the expression level of the cancer-associatedgene to be detected is high. It is also possible to determine theexpression level of the cancer-associated gene from an IOD of a band ona fluorescent image instead of a fluorescent intensity.

[0048] In order to carry out a more accurate determination, the degreeof amplification needs to be expressed numerically. For example, aquantitative PCR method (Japanese Unexamined Patent Publication No. Hei5-504886) may be applied in the step of nucleic acid amplificationreaction, whereby achieving the purpose mentioned above. A typicalmethod includes adding a known amount of a nucleic acid having at itsboth terminals the primer nucleotide sequences used in amplification ofa desired gene and having different internal sequences and sizes as aninternal standard and amplifying by PCR reaction; and deducing thedesired gene level by comparing the final amplified level of the desiredproduct in the light of the final amplified level of the internalstandard. In the present invention, an internal standard is not limitedto an externally added standard, and there may also be used cDNAobtained by using as a template mRNA of a gene expressing in a normaltissue and a cancer tissue in the same level. As such cDNA, for example,there can be included cDNA for β-actin gene which is a constituent of acell backbone.

[0049] For example, in RT-PCR method using a crude RNA sample extractedfrom gastric cancer tissue cells, when the synthetic oligonucleotideshaving the nucleotide sequences of SEQ ID NOs: 20 and 21 in SequenceListing are used as a primer pair for nucleic acid amplificationreaction, it is possible to only amplify the nucleotide sequence regionas shown in base numbers 122 to 487 in SEQ ID NO: 66 in Sequence Listingof the cDNA nucleotide sequences of a CA11 gene in the presentspecification as shown in FIG. 3(a).

[0050] The expression level of the cancer-associated gene can bedetermined by RNase protection assay by adding a probe which is RNA inan excess amount capable of specifically hybridizing with mRNA for acancer-associated gene to be detected or a partial portion thereof to agiven amount of a crude RNA sample extracted from a resected specimen,and quantifying the remaining RNA after digestion with the RNase. Inother words, the larger the amount of the remaining RNA, it can bedetermined that the expression level of the cancer-associated gene ishigh.

[0051] Incidentally, a probe used in this method is not particularlylimited, as long as it is RNA capable of hybridizing in hybridizationbuffer, for example, comprising 80% formamide, 40 mM Pipes (pH 6.4), 400mM NaCl and 1 mM EDTA at 45° C. for 20 hours, and having a nucleotidesequence complementary with a nucleotide sequence specific to mRNA for acancer-associated gene to be detected. In addition, the label on thisprobe is not particularly limited, and there may, for example, be used aradioactive substance typically exemplified by ³²P, or a fluorescentsubstance typically exemplified by fluorescein.

[0052] The expression level of the cancer-associated gene can bedetermined by Northern hybridization by fractionating a given amount ofa crude RNA sample extracted from a sample tissue based on the molecularweight; immobilizing on a nylon filter, or the like; bringing mRNA for acancer-associated gene to be detected into contact with an excess amountof a probe for detecting this gene, and determining the signal intensityobtained from the probe hybridizing with the immobilized RNA. In otherwords, the higher the signal intensity, it can be determined that theexpression level of the cancer-associated gene is high.

[0053] Incidentally, the term “hybridizing” used in the method refers,for example, to those capable of hybridizing by a process comprisingincubating at 42° C. for 20 hours in hybridization buffer containing 50%formamide, 0.65M NaCl, 0.1M sodium-Pipes, 5× Denhardt's reagent, 0.1%SDS, 5 mM EDTA. The detection probe is preferably a nucleic acid havinga nucleotide sequence complementary to a nucleotide sequence which isspecific to a cancer associated-gene mRNA to be detected. The nucleicacid is not particularly limited, as long as mRNA to be detected can beparticularized by location of the above signals, even if its nucleotidesequence is such that signals can be obtained at several spots in thedetection of RNA. Labelling of the above probe is not particularlylimited, and there can be used, for example, radioactive substancestypically exemplified by ³²P, as well as fluorescent substancestypically exemplified by fluorescein.

[0054]FIG. 2 shows one example of the change in the expression level ofmRNA for a cancer-associated gene detected by Northern hybridizationmethod. In this figure, a photograph of an autoradiogram obtained bysubjecting each of the RNAs obtained from a cancer tissue and a controlnormal tissue to electrophoresis individually, and hybridizing with alabeled probe for detecting mRNA for CA11 gene in the presentspecification.

[0055] In addition, when the change in the expression level of acancer-associated gene is confirmed using a protein as an index, theconfirmation may be made based on the biological activity of theprotein, and the detection using an antibody against the protein ispreferred for its simplicity in the present invention.

[0056] The antibody in the present invention is an antibody capable ofspecifically binding to a protein encoded by the cancer-associated gene.Therefore, the larger the amount of the antibody bound to a given amountof a crude protein extracted from a resected specimen, it can bedetermined that the expression level of the cancer-associated gene ishigh.

[0057] The protein as an antigen for obtaining the antibody describedabove can be obtained by purifying from cancer cells expressing thegene, or it can also be obtained by gene engineering technique. Forexample, a nucleic acid encoding the protein can be obtained by themethod described above, in which the DD method is combined with thescreening of the cDNA libraries prepared from cells expressing a desiredprotein. The desired protein can be obtained by incorporating the cDNAobtained into an appropriate expression vector, and expressing it in anappropriate host. Further, this protein may be expressed as a fusionprotein. For example, in order to increase the expression level of adesired protein, an appropriate peptide chain is added to the N-terminalor C-terminal derived from other proteins and then allowed to beexpressed, and a carrier having an affinity with this peptide chain isused, whereby a desired protein can be purified readily.

[0058] In addition, the antigen for obtaining an antibody may notnecessarily be an entire molecule of the protein, and the antigen may bea peptide having an amino acid sequence region which is capable ofrecognizing the antibody and specific to the protein.

[0059] As the method for obtaining an antibody, the antibody can, forexample, be obtained as an anti-serum by immunizing an animal with apeptide together with an adjuvant by a usual method. Alternatively, itcan be obtained as a monoclonal antibody according to the method ofGalfre, G. et al [Galfre, G. et al., Nature, 266, 550-552 (1977)].

[0060] An example of a method for detecting a protein using an antibodyincludes Western blotting method.

[0061] In this method, the method for detecting with a specific antibodycan be carried out by treating cells with a detergent to dissolveintracellular proteins; separating the protein by SDS-polyacrylamideelectrophoresis; transferring the resulting protein onto anitrocellulose membrane, and the like. The antibody bound to a proteincan secondarily be detected with, for instance, a ²⁵I-labeled protein A,a peroxidase-linked anti-IgG antibody, and the like.

[0062] The second invention of the present invention provides a kit fordetecting a cancer cell. In other words, there can be provided a kit fordetecting a cancer cell by utilizing the method for detecting a cancercell, which is the first invention of the present invention. Concretely,there can be exemplified a kit for detecting the change in theexpression level of a cancer-associated gene within the cells using asan index an amount of mRNA or an amount of a protein which is expressedby this gene.

[0063] In the case of a kit for detecting a cancer cell using as anindex an expression level of mRNA by using the detection method with thenucleic acid amplification described above in connection with the methodfor detecting a cancer cell, a primer pair is an essential constituent,where the primer pair has the characteristics described above inconnection with the method for detecting a cancer cell wherein theprimer pair is capable of detecting mRNA of which cDNA is DNA as shownin any one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing, orDNA capable of hybridizing under stringent conditions with DNA as shownin a nucleotide sequence comprising the nucleotide sequence as shown inany one of SEQ ID NOs: 1 to 16 and 66 to 68 in Sequence Listing. Forexample, the kit in the present invention utilizing RT-PCR as adetection method may comprise in addition to the primer pair describedabove reverse transcriptase, dNTPs and a thermostable DNA polymerase.Incidentally, the kinds and the number of the cancer-associated genes tobe detected by this kit are not particularly limited. Therefore, theprimer pair constituting this kit is not particularly limited, and itmay be selected appropriately depending upon the kinds and the number ofthe cancer-associated genes to be detected.

[0064] One example of the primer pair using as a template cDNA for thecancer-associated gene of the present invention only a part of theregion of which is specifically amplified is shown in Table 2. In eachprimer pair in the table, a symbol of a combination of an alphabet andnumerals indicates the name of the primer in the present invention, anda number within a parenthesis attached to each symbol indicates SEQ IDNO: in Sequence Listing showing the nucleotide sequence of each primer.Incidentally, β-actin shown in Table 2 is a gene selected as an internalstandard for the purpose of quantifying mRNA for the cancer-associatedgene in a crude RNA sample extracted from a resected specimen. TABLE 2Target Size of Amplified Gene Primer Pair DNA Predicted CA11 F1 (20) R1(21) 366 bp CA13 F2 (22) R2 (23) 168 bp CC24 F3 (24) R3 (25) 259 bp GG24F4 (26) R4 (27) 384 bp AG26 F5 (28) R5 (29) 389 bp GC31 F6 (30) R6 (31)213 bp GC32 F7 (32) R7 (33) 251 bp GC33 F8 (34) R8 (35) 563 bp GG33 F9(36) R9 (37) 218 bp CC34 F10 (38) R10 (39) 241 bp GC35 F11 (40) R11 (41)157 bp GC36 F12 (42) R12 (43)  95 bp CA42 F13 (44) R13 (45) 245 bp CC62F14 (46) R14 (47) 134 bp β-Actin F15 (48) R15 (49) 264 bp

[0065] On the other hand, in the case of a kit for detecting a cancercell using as an index mRNA by using a detection method employing RNaseprotection assay or Northern hybridization method, it is an essentialrequirement for a constituent to have a probe which has thecharacteristics described above in connection with the method fordetecting a cancer and is capable of detecting mRNA of acancer-associated gene, of which cDNA is DNA comprising the nucleotidesequence as shown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 inSequence Listing, or DNA capable of hybridizing under stringentconditions with DNA as shown in any one of SEQ ID NOs: 1 to 16 and 66 to68. For example, in the case of a kit utilizing RNase protection assay,the kit may comprise, in addition to the probe described above, RNase, aconcentrated reaction mixture for RNase, and the like. The kinds and thenumber of the cancer-associated genes to be detected by this kit are notparticularly limited. Therefore, a probe constituting this kit is notparticularly limited, and it may be selected appropriately depending onthe kinds and the number of the cancer-associated genes to be detected.

[0066] On the other hand, in the case of a kit for detecting a cancercell using a protein as an index by using the detection method with anantibody, it is an essential constituent to have an antibody which hasthe characteristics described above in connection with the method fordetecting a cancer cell and is capable of binding individually andspecifically to a peptide encoded by DNA as shown in any one of SEQ IDNOs: 1 to 16 and 66 to 68 in Sequence Listing, or DNA as shown in anucleotide sequence comprising a nucleotide sequence of DNA capable ofhybridizing under stringent conditions with DNA as shown in a nucleotidesequence comprising the nucleotide sequence as shown in any one of SEQID NOs: 1 to 16 and 66 to 68. The kinds and the number of thecancer-associated genes to be detected by this kit are not particularlylimited. Therefore, the antibody constituting this kit is notparticularly limited, and it may be selected appropriately dependingupon the kinds and the number of the cancer-associated genes to bedetected.

[0067] By using such a kit, a cancer cell can be detected more simply.Therefore, it is possible to diagnose a cancer based on the determinedexpression level of a cancer-associated gene by using such a kit. Inother words, humans whose confirmation of the presence of the cancercells is made by the method for detecting a cancer cell using this kitcan be determined to be cancer-positive.

[0068] The third invention of the present invention is a method forcontrolling proliferation of a cancer cell using a substancespecifically binding to a cancer-associated gene or an expressionproduct thereof. The specific binding substance referred in the presentspecification can, for example, include nucleic acids, antibodies,cytotoxic T lymphocytes (CTL), and the like.

[0069] For example, bcr-abl chimeric protein detected frequently inchronic myelocytic leukemia has a high tyrosine kinase activity andplays an important role in the onset and the proliferation of theleukemia. An antisense oligonucleotide against a gene encoding thischimeric protein can serve to suppress in vivo the proliferation of thisgene-expressing tumor (Skorski, T., Proc. Natl. Acad. Sci. USA 91, 4504,1994). On the other hand, a peptide peculiar to a cancer of a proteinexpressing specifically in a cancer cell has been conventionally knownto be a target of T cell immunoresponse to a cancer cell, and a peptidein a proximal site of the fusion of this fusion protein is immunized,whereby obtaining T cells reactive with this fusion protein (Chen, W.,Proc. Natl. Acad. Sci. USA 89, 1468, 1992), which can, for example, becarried out utilizing the techniques described in the following report.Concretely, CD4+T cells which react specifically with a peptide for rasin which a 12th amino acid glycine is substituted with another aminoacid, and which have HLA-DR restrainability are separated in human Tcells (Jung, S., J. Exp. Med., 173, 273, 1991); and from a mouseimmunized with a recombinant vaccinia virus capable of producing aprotein for ras having a mutation in a 61st amino acid a CTL against apeptide consisting of 8 amino acids including such a mutation site canbe induced (Skipper, J., J. Exp. Med., 177, 1493, 1993). Further, in amouse immunized with a solubilized mutated protein for ras prepared by agene recombination, the proliferation of cancer cells having the samemutation in vivo is suppressed (Fenton, R. G., J. Natl. Cancer Inst.,85, 1294, 1993); and from spleen cells sensitized with a mutated peptidefor ras, a CTL exhibiting a cytotoxic activity on cancer cellsexpressing the same mutated ras is obtained (Peace, D. J., J. Exp. Med.,179, 473, 1994).

[0070] Therefore, as to a gene found to be associated with cancerationof cells in the present invention, it is possible to control the cellproliferation by using the same antisense oligonucleotide. In addition,if there can be obtained T cells reactive with a protein encoded by agene of which expression level is considered to be increased owing tocanceration, it is possible to suppress the proliferation of cells inwhich the protein is expressed at a high level.

[0071] The fourth invention of the present invention provides a novelpeptide usable for the detection of cancer, and a nucleic acid encodingthe above peptide. In the cancer associated-gene elucidated by thepresent inventors, genes except for CA11, CA13, GG33, GC35, GC36 andCA42 have been clarified as genes which have already been isolated andidentified by homology search with database in which information ofnucleotide sequences is recorded. Specifically, CC24 corresponds tocytochrome c oxidase subunit I gene [Horai, S. et al., Proc. Natl. Acad.Sci. USA 92, 532-536 (1995)]; AG26 corresponds to p190-B gene [Burbelo,P. D. et al., J. Biol. Chem. 270, 30919-30926 (1995)]; GC31 correspondsto cytochrome c oxidase subunit II gene [Power, M. D. et al., NucleicAcids Res. 17, 6734 (1989)]; GC32 corresponds to cytochrome b gene[Anderson, S. et al., Nature 290, 457-465 (1981)]; GC33 corresponds tointegrin α 6 subunit gene [Tamura, R. N. et al., Journal of CellBiology, 111, 1593-1604 (1990)]; GG24 corresponds to F1-ATPase β subunitgene [Ohta, S. et al., The Journal of Biochemistry, 99, 135-141 (1986)];and CC62 corresponds to lactoferrin gene [Rey, M. W. et al., NucleicAcids Res. 18, 5288 (1990)]. On the other hand, CC34 cDNA clone is aclone different from a partial region of the cDNA nucleotide sequenceencoding 16SrRNA [Horai, S. et al., Proc. Natl. Acad. Sci. USA 92,532-536 (1995)] by 7 bases. Incidentally, the association withcarcinogenesis for these genes has not been known.

[0072] On the other hand, as to each of the genes of CA11, CA13, GG33,GC35 and CA42, no reports have been yet made with regard to thenucleotide sequence, the sequence identical to the amino acid sequenceencoded therein or the sequence having a homology therewith in theregion analyzed herein in each of cDNAs for the genes. As a result ofadditional analysis, it is clarified that a nucleotide sequence of cDNAfor GC36 gene has homology with a nucleotide sequence of cDNA for nCL-4as mentioned above. Here, the cDNA for nCL-4 has a nucleotide sequence,wherein 78 bp of bases are inserted between base numbers 956 and 957 ofSEQ ID NO: 68 in Sequence Listing, and 241 bp at 3′-terminal of basesare deleted. Namely, GC36 cDNA sequence is different from nCL-4 cDNAsequence. In other words, in the nucleotide sequence of each of cDNAsfor the genes of CA11, CA13, GG33, GC35, GC36 and CA42, a nucleic acidhaving the nucleotide sequence clarified in the present invention is anovel nucleic acid isolated for the first time by the present inventors.

[0073] As shown in Table 1, a peptide encoded by a novel nucleic acid inthe present invention comprising the nucleotide sequence as shown ineach of SEQ ID NOs: 66, 2, 13 and 68 in Sequence Listing is deducedbased on this nucleotide sequence that the peptide comprises the aminoacid sequence as shown in each of SEQ ID NOs: 69, 18, 19 and 70 inSequence Listing, without being limited thereto. Specifically, therealso are encompassed [1] a peptide comprising an entire portion of theamino acid sequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and70 in Sequence Listing, or a partial portion thereof; and [2] a peptideresulting from addition, deletion or substitution of one or more aminoacids in the amino acid sequence as shown in any one of SEQ ID NOs: 17to 19, 69 and 70 in Sequence Listing, and having a change in theexpression level owing to canceration of cells, because of the reasonsdescribed below.

[0074] In a naturally-occurring protein, mutations such as deletion,insertion, addition and substitution of amino acids can take place inits amino acid sequence in addition to a polymorphism or a mutation in agene encoding it as well as a modification in vivo or in purificationstep after its production. Nevertheless, when such a mutation is presentin a region in which it is insignificant to preserve the activities andthe structure of the protein, there have been known to exhibitphysiological and biological activities substantially of the same levelas those of the proteins without mutations.

[0075] In addition, the same can be said for the case where themutations described above are artificially introduced into an amino acidsequence of the protein, in which case diversified, various kinds ofmutants can be further prepared. For instance, it has been also knownthat a polypeptide resulting from substitution of a particular cysteineresidue with serine in the amino acid sequence of human interleukin 2(IL-2) retains IL-2 activity [Wang, A. et. al., Science, 224, 1431-1433(1984)]. Therefore, proteins are encompassed within the scope of thepresent invention, as long as no difference in the change in anexpression level owing to canceration is found, even if the protein hasan amino acid sequence which results from deletion, insertion, additionor substitution of one or several amino acid residues in an amino acidsequence disclosed by the present invention.

[0076] Further, certain kinds of proteins have been known to have apeptide region which is unessential for its activity. Examples aresignal peptide present in a protein secreted extracellularly, and apro-sequence found in a precursor of a protease, or the like, and almostall of these regions are removed after translation or when convertedinto an active protein. Such proteins are present in the form ofdifferent primary structures, but the proteins exhibit equivalentfunctions eventually.

[0077] When a protein is produced by a gene engineering technique, apeptide chain irrelevant to the activity of a, desired protein may beadded to an amino terminal or carboxyl terminal of the protein. Forexample, in order to increase the expression level of a desired protein,a fusion protein resulting from adding a part of an amino terminalregion of a protein highly expressed in a host used to an amino terminalof a desired protein may be prepared. Alternatively, in order tofacilitate the purification of the protein expressed, a peptide havingan affinity with a particular substance may be added to an aminoterminal or carboxyl terminal of a desired protein. These added peptidesmay remain in an added state when there is no adverse effect on theactivity of a desired protein, or the added peptides may be removed froma desired protein, if necessary, by means of an appropriate treatmentsuch as a limited degradation with a protease.

[0078] Even a protein having or adding a peptide unessential for itsfunction is also encompassed within the scope of the protein of thepresent invention, as long as it can exhibit an equivalent function. Theterm “peptide” in the present specification refers to two or more aminoacids bound to each other via peptide bonds, and is intended toencompass those referred to as “protein.”

[0079] A partial portion of the novel nucleic acid in the presentinvention consists of a nucleic acid encoding a peptide having the aminoacid sequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and 70 inSequence Listing, wherein its nucleotide sequence include those as shownin Table 1, for instance, the nucleotide sequence as shown in any one ofSEQ ID NOs: 1, 2, 13, 66 and 68 and in Sequence Listing. In other words,the peptide having the amino acid sequence as shown in SEQ ID NO: 17 inSequence Listing is encoded by the base numbers 2 to 598 of thenucleotide sequence as shown in SEQ ID NO: 1 in Sequence Listing; thepeptide having the amino acid sequence as shown in SEQ ID NO: 69 inSequence Listing is encoded by the base numbers 64 to 660 of thenucleotide sequence as shown in SEQ ID NO: 66 in Sequence Listing; thepeptide having the amino acid sequence as shown in SEQ ID NO: 18 inSequence Listing is encoded by the base numbers 1698 to 1850 of thenucleotide sequence as shown in SEQ ID NO: 2 in Sequence Listing; thepeptide having the amino acid sequence as shown in SEQ ID NO: 70 inSequence Listing is encoded by base numbers 83 to 2074 of the nucleotidesequence as shown in SEQ ID NO: 68; the peptide having the amino acidsequence as shown in SEQ ID NO: 19 in Sequence Listing is encoded by thebase numbers 8 to 196 of the nucleotide sequence as shown in SEQ ID NO:13 in Sequence Listing, respectively, but the nucleic acids encoding thenovel peptide in the present invention are not limited thereto.Specifically, there are also encompassed within the present invention 1)a nucleic acid encoding a peptide usable for detection of a cancer cell,wherein the peptide comprises an entire sequence of the amino acidsequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and 70 inSequence Listing, or a partial sequence thereof; 2) a nucleic acidencoding a peptide capable of changing its expression level owing tocanceration of a cell, wherein the nucleic acid is capable ofhybridizing with the novel nucleic acid of the present invention understringent conditions; 3) a nucleic acid encoding a peptide usable fordetection of a cancer cell by the change in its expression level,wherein one or more amino acids are added, deleted or substituted in theamino acid sequence as shown in any one of SEQ ID NOs: 17 to 19, 69 and70 in Sequence Listing, and the like.

[0080] The term “nucleic acid encoding an amino acid sequence” describedin the present specification will be described. There has been knownthat as the codon (triplet base combination) designating a particularamino acid on a gene, 1 to 6 kinds each exist for every amino acid.Therefore, there can be a large number of nucleic acids each encoding anamino acid sequence, depending on its amino acid sequence. In nature,the gene does not exist in a stable form, and it is not rare that themutation of its nucleotide sequence takes place. The mutation on thegene may not affect the amino acid sequence to be encoded (so-called“silent mutation”), in which case it can be said that different nucleicacids encoding the same amino acid sequence have been produced. Therecannot, therefore, be denied the possibility that even when the nucleicacid encoding a particular amino acid sequence is isolated, a variety ofnucleic acids encoding the same amino acid sequence are produced withgeneration passage of the organism containing them. Moreover, it is notdifficult to artificially produce a variety of the nucleic acidsencoding the same amino acid sequence by means of various geneticengineering techniques. For example, when a codon used on a naturalnucleic acid encoding the desired protein is low in usage in the host inthe production of a protein by genetic engineering, the expression levelof the protein is sometimes insufficient. In such a case, highexpression of the desired protein is achieved by artificially convertingthe codon into another one of commonly used in the host without changingthe amino acid sequence encoded (for example, Japanese Examined PatentPublication No. Hei 7-102146). It is of course possible to artificiallyproduce a variety of nucleic acids encoding a particular amino acidsequence, and the nucleic acids can be also produced in nature.Therefore, the present invention includes a nucleic acid, as long as thenucleic acid encodes an amino acid sequence disclosed in the presentspecification, even if it is not a nucleic acid having same nucleotidesequence disclosed in the present specification.

[0081] In fact, in the present invention, nucleic acids of whichnucleotide sequences are slightly different but the amino acid sequenceencoded is identical is obtained. Although R at base number 1784 is A,and K at base number 1985 is T in the nucleotide sequence as shown inSEQ ID NO: 2 in Sequence Listing of which the nucleotide sequence iscontained in a nucleotide sequence for cDNA of CA13 gene, there isobtained cDNA in which R at base number 1784 is G, and K at base number1985 is T; and a nucleic acid in which R at base number 1784 is A, and Kat base number 1985 is G in the nucleotide sequence as shown in SEQ IDNO: 2 in Sequence Listing. However, the differences of the nucleotidesequence at these two sites do not affect the amino acid sequenceencoded in base numbers 1698 to 1850 in the nucleotide sequence as shownin SEQ ID NO: 2 in Sequence Listing, and each peptide encoded by theabove three kinds of nucleic acids has the amino acid sequence as shownin SEQ ID NO: 18 in Sequence Listing.

[0082] Among the cDNAs for novel genes of the present invention, cDNAfor CA11 gene has the nucleotide sequence as shown in SEQ ID NOs: 1 and66; cDNA for CA13 gene has the nucleotide sequence as shown in SEQ IDNO: 2; cDNA for GG33 gene has the nucleotide sequence as shown in SEQ IDNO: 9; cDNA for GC35 gene has the nucleotide sequences as shown in SEQID NOs: 11 and 67; cDNA for GC36 gene has the nucleotide sequences asshown in SEQ ID NOs: 12, 15, 16 and 68; and cDNA for CA42 gene has thenucleotide sequences as shown in SEQ ID NO: 13. Here, the nucleotidesequence as shown in SEQ ID NO: 66 comprises the nucleotide sequences asshown in SEQ ID NO: 1; the nucleotide sequence as shown in SEQ ID NO: 67comprises the nucleotide sequences as shown in SEQ ID NO: 11; and thenucleotide sequence as shown in SEQ ID NO: 68 comprises the nucleotidesequences as shown in SEQ ID NOs: 12, 15 and 16.

[0083] Moreover, the novel nucleic acids of the present inventioninclude a nucleic acid capable of hybridizing with the nucleic acidhaving the nucleotide sequences as shown in any one of SEQ ID NOs: 66,2, 9, 67, 13 as well as 68 in Sequence Listing under stringentconditions, wherein the nucleic acid is complementary to a nucleotidesequence for mRNA capable of changing an expression level bycanceration. In fact, the nucleic acid having the above properties isobtained in the present invention. For instance, there are obtained theabove nucleic acid of which nucleotide sequence is slightly differentbut an encoded amino acid sequence is identical.

[0084] In addition, the fifth invention of the present inventionprovides an antibody against the peptide encoded by the novel nucleicacid in the present invention. The above antibody can be utilized fordetection of the cancer cell described above.

EXAMPLES

[0085] The present invention will be described more concretelyhereinbelow by means of the working examples, without intending torestrict the scope of the present invention thereto.

Example 1 Analysis of Cancer-Associated Gene

[0086] 1) Confirmation of mRNA Which can Serve as Index for DetectingCancer

[0087] There was confirmed whether or not mRNA of which expression levelwas changed by canceration was present by DD method comprising comparingthe expression of mRNA of a cancerated lesion tissue with that of acontrol normal tissue of a stomach as detailed below.

[0088] First, from each of a cancer tissue and a control normal tissueof a stomach excised from a patient with an advanced,poorly-differentiated adenocarcinoma, RNA was extracted with TRIzol™reagent (manufactured by Gibco BRL) to obtain a crude RNA sample. A 50μg portion of the crude RNA sample thus obtained was reacted with 10units of DNaseI (manufactured by Takara Shuzo Co., Ltd.) at 37° C. for30 minutes in the presence of 5 mM MgCl₂ as a final concentration and 20units of RNase inhibitor (manufactured by Takara Shuzo Co., Ltd.) toremove genomic DNA, Using this RNA, RT-PCR was carried out withDifferential Display™ Kit (manufactured by Display Systems) and EnzymeSet-DD (manufactured by Takara Shuzo Co., Ltd.) in accordance with theprocedures described in the instruction attached to the kit.

[0089] Specifically, reverse transcription reaction was carried out perone reaction by mixing 200 ng of the crude RNA sample treated with theabove DNase, and any one kind of the oligonucleotides having thenucleotide sequences as shown in SEQ ID NOs: 56 to 64 in SequenceListing as a primer, thereafter heat-treating at 70° C. for 10 minutes,subjecting to rapid cooling, and subsequently reacting with AMV reversetranscriptase at 55° C. for 30 minutes. Other downstream primers wereindividually reacted in the same manner to prepare 9 kinds ofsingle-stranded cDNA samples in total.

[0090] In the subsequent nucleic acid amplification reaction by PCR, anucleic acid amplification was carried out by PCR using each of the 9kinds of single-stranded cDNAs described above as a template, anoligo(dT) primer identical to that used in the reverse transcription asa downstream primer, and any one kind of the 10mer-oligonucleotides inthe kit which had the nucleotide sequences as shown in SEQ ID NOs: 50 to55 in Sequence Listing as an upstream primer, to prepare 54 kinds ofamplified DNA samples in total.

[0091] The PCR was carried out by adding 3 mM MgCl₂, 15 μM each of dATP,dGTP, dCTP and dTTP as substrates, and 1.85 kBq/ml [α-³³P]-dATP(manufactured by Amersham) as a labelling compound, and reacting for 40cycles, wherein one cycle consists of at 94° C. for 30 seconds, at 40°C. for 60 seconds and at 72° C. for 60 seconds. After termination of thereaction, an equivolume of 95% formamide was added, and the mixture wassubjected to thermal denaturation at 90° C. for 2 minutes to obtain asample for electrophoresis. The electrophoresis was carried out on a 7 Murea-denatured 5% polyacrylamide gel, and autoradiography yielded afingerprint comprising a large number of bands, wherein there were foundto be bands having different signal intensities between theautoradiogram of the cancer tissue and that of the control normaltissue.

[0092] As one example, the results where D4 having the nucleotidesequence as shown in SEQ ID NO: 59 in Sequence Listing was used as adownstream primer, and U1 having the nucleotide sequence as shown in SEQID NO: 50 was used as an upstream primer are shown in FIG. 1.Specifically, FIG. 1 is a reproduced photograph of an autoradiogramshowing electrophoretic patterns of the DNA fragment obtained when acancer-associated gene was detected by the DD method. Here, in FIG. 1,1N is a lane wherein on an acrylamide gel was electrophoresed anamplified DNA fragment obtained by using as a template a crude RNAsample obtained from a normal tissue of a patient with apoorly-differentiated adenocarcinoma-type gastric cancer; and 1T is alane wherein on an acrylamide gel was electrophoresed an amplified DNAfragment obtained by using as a template a crude RNA sample obtainedfrom a cancer tissue of the same patient with the poorly-differentiatedadenocarcinoma-type gastric cancer, respectively. A band having astronger signal intensity in the autoradiogram obtained from the controlnormal tissue than in the autoradiogram of the cancer tissue sample wasfound at the position corresponding to about 750 bp as indicated with“→” in FIG. 1. The present inventors named the gene expressing the mRNAwhich causes the band to show this difference in the intensity as CA11.

[0093] Table 3 showed the combination of the upstream and downstreamprimers for detecting the difference in the expression level of eachmRNAs by means of the DD method, an the approximate size of an amplifiedDNA fragment, and the difference in the level of the amplified DNAobtained by RT-PCR from the cancer tissue and the control normal tissuefor each of genes which was detected by the present inventors with theDD method and named. In the column of the primers in Table 3, a symbolof a combination of an alphabet and numerals indicates the name of aprimer, and a number within a parenthesis attached to each symbolindicates SEQ ID NO: showing the nucleotide sequence of the primer inSequence Listing. TABLE 3 Approximate Size of Difference in Name ofPrimer Pair Amplified Amount of Gene Upstream Downstream DNA fragmentDNA fragment CA11 U1 (50) D4 (59) 750 bp Cancer Tissue < Normal TissueCA13 U1 (50) D4 (59) 620 bp Cancer Tissue > Normal Tissue CC24 U2 (51)D5 (60) 800 bp Cancer Tissue > Normal Tissue GG24 U2 (51) D9 (61) 480 bpCancer Tissue > Normal Tissue AG26 U2 (51) D3 (58) 550 bp Cancer Tissue< Normal Tissue GC31 U3 (52) D8 (63) 440 bp Cancer Tissue > NormalTissue GC32 U3 (52) D8 (63) 310 bp Cancer Tissue > Normal Tissue GC33 U3(52) D8 (63) 300 bp Cancer Tissue > Normal Tissue GG33 U3 (52) D9 (64)410 bp Cancer Tissue > Normal Tissue CC34 U3 (52) D5 (60) 290 bp CancerTissue > Normal Tissue GC35 U3 (52) D8 (63) 210 bp Cancer Tissue <Normal Tissue GC36 U3 (52) D8 (63) 190 bp Cancer Tissue < Normal TissueCA42 U4 (53) D4 (59) 660 bp Cancer Tissue > Normal Tissue CC62 UG (55)D5 (60) 380 bp Cancer Tissue < Normal Tissue

[0094] 2) Identification of mRNA Serving as Index for Detecting Cancer

[0095] There was investigated whether a change in an expression level ofthe mRNA used as a template for an amplified DNA fragment derived fromeach of the genes shown in Table 3 as confirmed by the DD method inSection 1) described above was truly associated with canceration.

[0096] First, the studies were made by means of Northern hybridization.Specifically, there was studied whether the difference in the expressionlevels of the mRNA of a cancer-associated gene expressed in a cancertissue and that in a control normal tissue could be detected by usingeach amplified DNA fragment obtained by the method in Section 1)described above as a probe.

[0097] The probe for the detection was prepared as follows.Specifically, from the acrylamide gel on which the amplified DNAfragment obtained by the DD method in Section 1) described above waselectrophoresed, the region containing each amplified DNA fragment shownin Table 3 was cut out, and thereto was added 100 μl of water andsubjected to a heat extraction to collect individually each DNA fragmentcontained. Re-amplification by PCR was carried out by using each DNAfragment individually as a template, with a combination of the upstreamand downstream primers used to obtain each DNA fragment shown in Table3. Further, about 100 ng of each amplified DNA fragment was labeled with³²P using Random Primer DNA Labeling Kit (manufactured by Takara ShuzoCo., Ltd.) to prepare 14 kinds of probes for detection. Separately fromabove, mRNA for β-actin gene was selected as a positive control of acrude RNA extracted from each tissue, and the synthetic oligonucleotidehaving the nucleotide sequence as shown in SEQ ID NO: 65 in SequenceListing was labeled in the same manner with ³²P to obtain a probe fordetecting mRNA for β-actin gene. Thereafter, the probe for detectiondescribed above was mixed together with herring sperm DNA so as to havea concentration of 100 μg/ml, and then heat-denatured. To the resultingreaction mixture was added hybridization buffer (50% formamide, 0.65 MNaCl, 0.1M Na-Pipes, 5× Denhardt's reagent, 0.1% SDS, 5 mM EDTA) toprepare 15 kinds of probe solutions for detection in Northernhybridization.

[0098] Northern hybridization was carried out as follows. First, 20 μgper well of a crude RNA sample extracted from each of a cancer tissueand a control normal tissue from the patient with a gastric cancerprepared as described above was subjected to electrophoresis on aformalin-denatured 1% agarose gel and blotted on a Hybond N⁺ membrane(manufactured by Amersham). Subsequently, a blotted membrane andhybridization buffer added with heat-denatured herring sperm DNA so asto have final concentration of 100 μg/ml were added to a Hybri Bag(manufactured by COSMO BIO). The resulting composition was allowed tostand at 42° C. for 2 hours, and then the buffer was discarded toprepare a membrane with pre-hybridization treatment. After preparing 15such membranes as above, to each membrane was added each of the 15 kindsof detection probe solutions for Northern hybridization described above,and hybridization was carried out at 42° C. for 16 hours. Thereafter,each blotted membrane was taken from the Hybri Bag, and rinsed withwashing solution I (2×SSC, 0.2% sodium pyrophosphate, 0.1% SDS) at 42°C. for 20 minutes, and then with washing solution II (0.5×SSC, 0.2%sodium pyrophosphate, 0.1% SDS) at 42° C. for 20 minutes. Incidentally,rinsing with washing solution II was repeated twice with replacing thewashing solution. The membrane after rinsing was wrapped with a plasticfilm and exposed for one day and night to a high-sensitivity X-ray film(manufactured by Kodak). From the signal intensity in the resultantautoradiogram, the expression level in the cancer tissue was comparedwith that of the control normal tissue.

[0099] As one example, the results of the detection of mRNA for CA11gene are shown in FIG. 2. In FIG. 2, 1N is a lane wherein on an agarosegel was electrophoresed a crude RNA sample obtained from a normal tissueof a patient with a poorly-differentiated adenocarcinoma-type gastriccancer; and 1T is a lane wherein on an agarose gel was electrophoresed acrude RNA sample obtained from a cancer tissue of the same patient withthe poorly-differentiated adenocarcinoma-type gastric cancer. (a) showsresults obtained with a probe for detecting CA11, and (b) shows resultsobtained with a probe for detecting β-actin. Since both of the 1N andthe 1T exhibited the signals obtained with the probes for detectingβ-actin as shown in (b), it is clear that in the both samples the RNA isextracted without undergoing degradation excessively. On the other hand,a clear signal as indicated by “→” at a position near 1.1 kb was presentonly in lane 1N but no signals were present in lane 1T as shown in (a).Therefore, it was found that the CA11 was a gene of which expressionlevel was reduced owing to canceration. Similarly, CC62 exhibited a bandat about 2.6 kb only on the autoradiogram derived from the controlnormal stomach tissue. GC31, GC32 and CC34 showed the bands at about 1.0kb, about 1.6 kb and about 1.7 kb, respectively, and in any of thesegenes more intensive signal was obtained for the crude RNA samplesprepared from the gastric cancer tissues as compared to that of thecrude RNA samples prepared from the control normal stomach tissues.Incidentally, the signal intensity was determined by measuring each bandof an autoradiogram by a densitometer. Subsequently, IOD of each bandobtained on the autoradiogram was calculated with FMBIO-100(manufactured by Hitachi Soft Engineering), and an index was calculatedby the equation as shown below to determine whether or not a gene was acancer-associated gene.

[Index Value]=(X×βY)/(Y×βX)  Equation 2:

[0100] In the above equation, each symbol expresses the following value:

[0101] X: IOD of a band derived from mRNA for the gene shown in Table 3obtained from a gastric cancer tissue;

[0102] Y: IOD of a band derived from mRNA for the gene shown in Table 3obtained from a control normal stomach tissue;

[0103] βX: IOD of a band derived from mRNA for β-actin gene obtainedfrom a gastric cancer tissue; and

[0104] βY: IOD of a band derived from mRNA for β-actin gene obtainedfrom a control normal stomach tissue.

[0105] The comparison on the expression level was made by carrying outRT-PCR with respect to each of the genes CA13, CC24, GG24, AG26, GC33,GG33, GC35, GC36 and CA42 in which no signals were obtained by Northernhybridization. In order to design a primer for the nucleic acidamplification reaction in the RT-PCR, each DNA fragment used as a probein Northern hybridization was subjected to a direct sequencing by PCR,or was cloned by a TA cloning procedure and then sequenced by a dideoxymethod, whereby determining its nucleotide sequence. The nucleotidesequences of primers designed based on the resulting nucleotide sequenceinformation and used in the RT-PCR with mRNA derived from each of thegenes as a template are as shown in any of SEQ ID NOs: 22 to 29, 34 to37 and 40 to 45 in Sequence Listing. Table 2 shows the genes togetherwith the corresponding primers used to confirm their expression.

[0106] A change in an expression level of mRNA by RT-PCR was confirmedby a DNaseI treatment of a crude RNA sample obtained from each of thecancer tissue and the control normal tissue of a patient with a gastriccancer prepared by the method in Section 1) described above. Thereafter,RT-PCR was carried out in a 100 μl reaction system of 40 ng of eachtreated sample with TaKaRa RNA PCR Kit Ver. 2.1 according to theprocedures described in the instruction attached to the kit.Specifically, 40 ng of a crude RNA sample as a template and an oligo(dT)primer (final concentration: 2.5 μM) as a downstream primer were used toprepare a reverse transcription reaction mixture (10 mM Tris-HCl, pH8.3, 50 mM KCl, 5 mM MgCl₂, 1 mM each of dNTPs, 100 units of RNaseinhibitor, 25 units of AMV reverse transcriptase), and the reversetranscription reaction was carried out at 30° C. for 10 minutes, and at55° C. for 20 minutes and then at 95° C. for 5 minutes. Each 10 μl ofthe reverse transcription reaction mixture was added to each 40 μl of 10kinds of PCR reaction mixtures (final concentration: 10 mM Tris-HCl, pH8.3, 50 mM KCl, 2.5 mM MgCl₂, 1.25 units of TaKaRa Taq DNA polymerase)individually containing the primer pairs (0.2 μM) for detecting each ofthe mRNAs for the genes of CA13, CC24, GG24, AG26, GC33, GG33, GC35,GC36, CA42 and β-actin to make up a volume of 50 μl. One cycle after thepre-incubation at 94° C. for 2 minutes in PCR consisted of the step ofincubation at 94° C. for 30 seconds, at 55° C. for 60 seconds, and thenat 72° C. for 60 seconds. The amount of an amplified DNA product wasquantified by subjecting the amplified DNA product to agarose gelelectrophoresis, staining the gel with ethidium bromide, calculating theIOD of each band on the fluorescent image with FMBIO-100 to obtain anindex for determining whether or not a gene is a cancer-associated genefrom Equation 2 shown above.

[0107] The results of Northern hybridization method and RT-PCR describedabove, and the patterns of the changes in the expression owing to thecanceration of each of the genes evident from these results were shownin Table 4. In the column of the patterns of the changes in theexpression, a gene of which expression was amplified owing tocanceration was indicated with “↑” and a gene of which expression wassuppressed owing to canceration was indicated with “↓”. Specifically, itwas determined in Table 4 that a gene having an index value greater than1 is a gene of which expression level was increased owing tocanceration, and a gene having an index value less than 1 is a gene ofwhich expression level was reduced owing to canceration. As a result,there were clarified that the genes CA13, CC24, GG24, GC31, GC32, GC33,GG33, CC34 and CA42 were those of which expression levels were increasedowing to canceration, and the genes CA11, AG26, GC35, GC36 and CC62 werethose of which expression levels were reduced owing to canceration.TABLE 4 Method of Patterns of Name of Determining Changes in Gene IndexValue Index Value Expression CA11 0.036 A ↓ CA13 6.3 B ↑ CC24 2.0 B ↑GG24 2.8 B ↑ AG26 0.52 B ↓ GC31 3.1 A ↑ GC32 3.6 A ↑ GC33 2.3 B ↑ GG332.2 B ↑ CC34 15 A ↑ GC35 0.0046 B ↓ GC36 0.048 B ↓ CA42 1.9 B ↑ CC620.56 A ↓

[0108] (note) In the table, “A” represents a determination from theautoradiogram in Northern hybridization, and “B” represents adetermination based on the electrophoretic gel image of the amplifiedproduct by RT-PCR.

[0109] 3) Acquisition of Cancer-Associated Gene cDNA

[0110] A cDNA fragment of each of these cancer-associated genes was thencloned. First, a cDNA library was prepared by fractionating mRNA from acrude RNA sample derived from a cancer tissue or a normal tissue, whichwas prepared by the method described in Section 1) with mRNAPurification Kit (manufactured by Pharmacia) on an oligo(dT) column, andplating a phage and a host cell XLI-Blue MRF′ at a cell density of about40,000 plaques per rectangular plate in a 10 cm×14 cm plate using aZAP-cDNA synthesis kit (manufactured by Stratagene) according to theprotocols attached to the kit. Thereafter, phage particles weretransferred onto a Hybond N⁺ membrane, and screening was carried out bymeans of plaque hybridization using a probe identical to that used inNorthern hybridization described in Section 2), whereby finding aUni-ZAP XR clone containing a desired cDNA gene. This recombinantUni-ZAP XR clone was converted into a pBluescript phagemide by means ofan in vitro excision method. The nucleotide sequence of a DNA fragmentincorporated into this recombinant phagemide was determined with afluorescent DNA sequencer (manufactured by ABI). The nucleotidesequences obtained from connection of the nucleotide sequences of thecDNA fragments contained in the cDNA library by means of walking basedon the nucleotide sequence of the DNA fragment incorporated into thephagemide are shown in SEQ ID NOs: 2 to 10, 13, 14 and 68 in SequenceListing. Since cDNAs for CA11 and GC35 obtained above have smaller sizesof mRNA than the size deduced from the results of Northernhybridization, it is highly possible that 5′-terminal portion in each ofthe above cDNAs is deleted. Therefore, in order to obtain nearly a wholelength of cDNA, cDNA clones were isolated by again screening using acommercially available human gastric cDNA library (manufactured byTakara Shuzo Co., Ltd.) and a probe which was newly prepared based onproximal 5′-terminal region of the sequence obtained above. By means ofthe above screening, there were obtained a cDNA clone in which basenumbers 1 to 76 of SEQ ID NO: 66 in Sequence Listing were added to5′-terminal of the nucleotide sequence of SEQ ID NO: 1 in a case ofCA11; and a cDNA clone in which base numbers 1 to 2530 of SEQ ID NO: 67in Sequence Listing were added to 5′-terminal of the nucleotide sequenceof SEQ ID NO: 11 in Sequence Listing in a case of GC35.

[0111] Each of the nucleotide sequences thus obtained was subjected to ahomology search with known gene cDNA nucleotide sequences recorded inGenebank by using BLAST program [Altschul, S. F., Journal of MolecularBiology, 215, 403-410, (1990)]. As a result, there have not beenreported any sequences corresponding to the cDNA of each of CA11, CA13,GC36, GG33, GC35, GC36 and CA42, so that these genes were determined tobe novel genes. Further, as a result of searching an open reading framefor a gene product based on the nucleotide sequence contained in each ofthe gene cDNAs of CA11, CA13, GC36 and CA42, it was deduced that CA11cDNA encodes the amino acid sequence as shown in SEQ ID NO: 69 inSequence Listing, CA13 cDNA encodes the amino acid sequence as shown inSEQ ID NO: 18 in Sequence Listing, GC36 cDNA encodes the amino acidsequence as shown in SEQ ID NO: 70 in Sequence Listing, and CA42 cDNAencodes the amino acid sequence as shown in SEQ ID NO: 19 in SequenceListing, respectively. On the other hand, CC24 corresponded tocytochrome c oxidase subunit I gene, AG26 to p190-B gene, GC31 tocytochrome c oxidase subunit II gene, GC32 to cytochrome b gene, GC33 tointegrin a 6 subunit gene, GG24 to F1-ATPase β subunit gene, and CC62 tolactoferrin gene. Moreover, the nucleotide sequence region as shown inSEQ ID NO: 10 in Sequence Listing for the CC34 cDNA was found to bedifferent from a partial region of the cDNA encoding a mitochondrial16SrRNA by 7 bases.

[0112] Incidentally, in the screening of the cDNA library using as aprobe an amplified DNA fragment derived from CC34, in addition to thecDNA clone having the nucleotide sequence as shown in SEQ ID NO: 10 inSequence Listing, an additional, different kind of positive cDNA clonewas obtained. There was clarified that the nucleotide sequence of thiscDNA had a nucleotide sequence in which T at base number 935 in thenucleotide as shown in SEQ ID NO: 10 in Sequence Listing was substitutedwith A, and 6 bases consisting of GTTAAG at the 3′-terminal weredeleted, of which 1540 bases out of the entire 1546 bases of the entirenucleotide sequence had an identical sequence to a partial region of thecDNA encoding a mitochondrial 16SrRNA.

Example 2 Confirmation of Change in Gene Expression in Cancer Tissue

[0113] With respect to each cancer-associated gene confirmed in Example1, the association of the expression of this gene with the cancerationof cells was evaluated by using a cancer tissue different from that usedin Example 1.

[0114] 1) Confirmation of Change in Gene Expression in Cancer Tissue ofPatient with Signet Ring Cell Gastric Cancer

[0115] Using a crude RNA sample prepared in the same manner as inSection 1) of Example 1 from each of a cancer tissue and a controlnormal tissue excised from a patient with a signet ring cell gastriccancer who was different from the one provided the tissues used inSections 1) and 2) of Example 1, the expression levels in the cancertissue and the normal tissue were compared with respect to each of the14 kinds of cancer-associated genes clarified in Section 3) of Example 1by using the expression level of the mRNA as an index by means ofcarrying out Northern hybridization or RT-PCR described in Section 2) ofExample 1. As one example, the results of the detection of mRNA for CA11gene by RT-PCR method are shown in FIG. 3. Specifically, FIG. 3 is aphotograph of a fluorescent image of the electrophoresis of a DNAfragment obtained when a change in an expression level of acancer-associated gene is detected by RT-PCR method. The reactionconditions of the RT-PCR were according to the method described inSection 2) of Example 1, with setting two patterns in the number of thecycles of the PCR, i.e., 25 and 30. In FIG. 3, (a) shows the results ofthe detection of the expression of a cancer-associated gene CA11, and(b) shows the results of the confirmation of the expression of β-actinas a positive control. In FIG. 3, 2T is an amplified DNA fragmentobtained by using as a template a crude RNA sample extracted from agastric cancer tissue of the patient with a signet ring cell gastriccancer, and 2N is an amplified DNA fragment obtained by using as atemplate a crude RNA sample extracted from a normal gastric tissue ofthe patient with the signet ring cell gastric cancer. Also, the numerals“25” and “30” in FIG. 3 are the numbers of the cycles of the nucleicacid amplification in the RT-PCR method. Table 5 shows the results ofcalculated IODs of the bands on the fluorescent image shown in FIG. 3.Incidentally, each index shown in Table 5 was calculated from Equation 2described in Section 2) of Example 1. TABLE 5 Number of Cycles 25 30Sample Name 2T 2N 2T 2N CA11 365 31118 6345 61742 β-Actin 710 562 2511520425 Index Value 0.0093 0.083

[0116] In Table 5, since the IOD values of the band derived from β-actinobtained on the fluorescent image of 2T and 2N were of the similar levelin the PCR cycles of 25 and 30, there was clarified that RNAs could besimilarly extracted from all samples. However, since the index was lessthan 1 for both the 25 and 30 cycles of the PCR, there was clarifiedthat CA11 was a gene of which expression level was reduced owing tocanceration even also with patients with a signet ring cell gastriccancer. With respect to 13 kinds of cancer-associated genes other thanCA11, there was found to be a change in the expression level in the samemanner as in Section 2) of Example 1, so that there was clarified thatthe change in the expression level of each of the 14 kinds of genes asclarified in Section 3) of Example 1 was not a change peculiar to thetissue of the patient tested in Section 1) of Example 1.

Example 3

[0117] Construction of Kit for Detecting Cancer

[0118] A kit for detecting a cancer utilizing RT-PCR method comprisingthe following components was constructed.

[0119] Specifically, a kit comprises DNaseI, AMV reverse transcriptase,RNase inhibitor, 10×RT-PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl),25 mM MgCl₂, and a mixture of 2.5 mM each of dATP, dGTP, dCTP and dTTP,an oligo(dT) primer, Taq DNA polymerase, a primer pair specific to eachof the genes and a primer pair for amplifying β-actin gene as a positivecontrol shown in Table 2. In the column of the primer pair in Table 2, asymbol of a combination of an alphabet and a numeral indicates the nameof a primer, and a number within a parenthesis following each symbolindicates SEQ ID NO: showing the nucleotide sequence of the primer inSequence Listing.

[0120] According to the present invention, it is made possible to simplyand rapidly detect cancer. In addition, the presence of a novel nucleicacid associated with the cancer is elucidated.

Equivalent

[0121] Those skilled in the art will recognize, or be able to ascertainusing simple routine experimentation, many equivalents to the specificembodiments of the invention described in the present specification.Such equivalents are intended to be encompassed in the scope of thefollowing claims.

1 70 1 738 DNA Homo sapiens any n or Xaa = unknown 1 cctctgtccactgctttcgt gaagacaaga tgaagttcac aattgtcttt gctggacttc 60 ttggagtctttctagctcct gcccttgcta actataatat caacgtcaat gatgacaaca 120 acaatgctggaagtgggcag cagtcagtga gtgtcaacaa tgaacacaat gtggccaatg 180 ttgacaataacaacggatgg gactcctgga attccatctg ggattatgga aatggctttg 240 ctgcaaccagactctttcaa aagaagacat gcattgtgca caaaatgaac aaggaagtca 300 tgccctccattcaatccctt gatgcactgg tcaaggaaaa gaagcttcag ggtaagggac 360 caggaggaccacctcccaag ggcctgatgt actcagtcaa cccaaacaaa gtcgatgacc 420 tgagcaagttcggaaaaaac attgcaaaca tgtgtcgtgg gattccaaca tacatggctg 480 aggagatgcaagaggcaagc ctgttttttt actcaggaac gtgctacacg accagtgtac 540 tatggattgtggacatttcc ttctgtggag acacggtgga gaactaaaca attttttaaa 600 gccactatggatttagtcgt ctgaatatgc tgtgcagaaa aaatatgggc tccagtggtt 660 tttaccatgtcattctgaaa tttttctcta ctagttatgt ttgatttctt taagtttcaa 720 taaaatcatttagcattg 738 2 2042 DNA Homo sapiens any n or Xaa = unknown 2 ccgtgacaacactcctgtca tattggagtc caaaacttga attctgggtt gaatttttta 60 aaaatcaggtaccacttgat ttcatatggg aaattgaagc aggaaatatt gagggcttct 120 tgatcacagaaaactcagaa gagatagtaa tgctcaggac aggagcggca gccccagaac 180 aggccactcatttagaattc tagtgtttca aaacactttt gtgtgttgta tggtcaataa 240 catttttcattactgatggt gtcattcacc cattaggtaa acattccctt ttaaatgttt 300 gtttgttttttgagacagga tctcactctg ttgccagggc tgtagtgcag tggtgtgatc 360 atagctcactgcaacctcca cctcccaggc tcaagcctcc cgaatagctg ggactacagg 420 cgcacaccaccatccccggc taatttttgt attttttgta gagacggggt tttgccatgt 480 tgccaaggctggtttcaaac tcctggactc aagaaatcca cccacctcag cctcccaaag 540 tgctaggattacaggcatga gccactgcgc ccagccctta taaatttttg tatagacatt 600 cctttggttggaagaatatt tataggcaat acagtcaaag tttcaaaata gcatcacaca 660 aaacatgtttataaatgaac aggatgtaat gtacatagat gacattaaga aaatttgtat 720 gaaataatttagtcatcatg aaatatttag ttgtcatata aaaacccact gtttgagaat 780 gatgctactctgatctaatg aatgtgaacg tgtagatgtt ttgtgtgtat ttttttaaat 840 gaaaactcaaaataagacaa gtaatttgtt gataaatatt tttaaagata actcagcatg 900 tttgtaaagcaggatacatt ttactaaaag gttcattggt tccaatcaca gctcataggt 960 agagcaaagaaagggtggat ggattgaaaa gattagcntn tgtntcggtg gcaggttccc 1020 acntcgcaagcaattggaaa caaaantttn ggggagtttt attttgcatt ngggtgtgtt 1080 ttatgttaagcaaaacatan tttagaanca aatgaaaaag gcaattgaaa atcccagnta 1140 tttcacctagatggnatagc caccntgagc agaacttngt gatgnttcat tctgnggaat 1200 tttgtgcttnctactgtata gtgcatgtgg tgtaggttac tctaactggt tttgtngacg 1260 taaacatttaaagtgttata ttttttataa aaatgtttat ttttaatgat atgagaaaaa 1320 ttttgttaggccacaaaaac actgcactgt gaacatttta gaaaaggtat gtcagactgg 1380 gattaatgacagcatgattt tcaatgactg taaattgcga taaggaaatg tactgattgc 1440 caatacaccccaccctcatt acatcatcag gacttgaagc caagggttaa cccagcaagc 1500 tacaaagagggtgtgtcaca ctgaaactca atagttgagt ttggctgttg ttgcaggaaa 1560 atgattataactaaaagctc tctgatagtg cagagactta ccagaagaca caaggaattg 1620 tactgaagagctattacaat ccaaatattg ccgtttcata aatgtaataa gtaatactaa 1680 ttcacagagtattgtaaatg gtggatgaca aaagaaaatc tgctctgtgg aaagaaagaa 1740 ctgtctctaccagggtcaag agcatgaacg catcaataga aagractcgg ggaaacatcc 1800 catcaacaggactacacact tgtatataca ttcttgagaa cactgcaatg tgaaaatcac 1860 gtttgctatttataaacttg tccttagatt aatgtgtctg gacagattgt gggagtaagt 1920 gattcttctaagaattagat acttgtcact gcctatacct gcagctgaac tgaatggtac 1980 ttcgkatgttaatagttgtt ctgataaatc atgcaattaa aataaagtga tgcaacatct 2040 tg 2042 31539 DNA Homo sapiens any n or Xaa = unknown 3 atgttcgccg accgttgactattctctaca aaccacaaag acattggaac actataccta 60 ttattcggcg catgagctggagtcctaggc acagctctaa gcctccttat tcgagccgag 120 ctgggccagc caggcaaccttctaggtaac gaccacatct acaacgttat cgtcacagcc 180 catgcatttg taataatcttcttcatagta atacccatca taatcggagg ctttggcaac 240 tgactagttc ccctaataatcggtgccccc gatatggcgt tcccccgcat aaacaacata 300 agcttctgac tcttacctccctctctccta ctcctgctcg catctgctat agtagaggcc 360 ggagcaggaa caggttgaacagtctaccct cccttagcag ggaactactc ccaccctgga 420 gcctccgtag acctaaccatcttctcctta cacctagcag gtgtctcctc tatcttaggg 480 gccatcaatt tcatcacaacaattatcaat ataaaacccc ctgccataac ccaataccaa 540 acgcccctct tcgtctgatccgtcctaatc acagcagtcc tacttctcct atctctccca 600 gtcctagctg ctggcatcactatactacta acagaccgca acctcaacac caccttcttc 660 gaccccgccg gaggaggagaccccattcta taccaacacc tatcctgatt tttcggtcac 720 cctgaagttt atattcttatcctaccaggc ttcggaataa tctcccatat tgtaacttac 780 tactccggaa aaaaagaaccatttggatac ataggtatgg tctgagctat gatatcaatt 840 ggcttcctag ggtttatcgtgtgagcacac catatattta cagtaggaat agacgtagac 900 acacgagcat atttcacctccgctaccata atcatcgcta tccccaccgg cgtcaaagta 960 tttagctgac tcgccacactccacggaagc aatatgaaat gatctgctgc agtgctctga 1020 gccctaggat tcatctttcttttcaccgta ggtggcctga ctggcattgt attagcaaac 1080 tcatcactag acatcgtactacacgacacg tactacgttg tagctcactt ccactatgtc 1140 ctatcaatag gagctgtatttgccatcata ggaggcttca ttcactgatt tcccctattc 1200 tcaggctaca ccctagaccaaacctacgcc aaaatccatt tcgctatcat attcatcggc 1260 gtaaatctaa ctttcttcccacaacacttt ctcggcctat ccggaatgcc ccgacgttac 1320 tcggactacc ccgatgcatacaccacatga aatatcctat catctgtagg ctcattcatt 1380 tctctaacag cagtaatattaataattttc atgatttgag aagccttcgc ttcgaagcga 1440 aaagtcctaa tagtagaagaaccctccata aacctggagt gactatatgg atgcccccca 1500 ccctaccaca cattcgaagaacccgtatac ataaaatct 1539 4 1807 DNA Homo sapiens any n or Xaa = unknown4 gaattctttc ttcagcccat gtaaacatga aaataagggt taaaaatgac ttcattatgg 60ggaaaaggga caggatgcaa attgttcaaa ttccgggtgg ccgctgctcc ggcctccggg 120gccttgcgga gactcacccc ttcagcgtcg ctgcccccag ctcagctctt actgcgggcc 180gtccgacggc ggtcccatcc tgtcagggac tatgcggcgc aaacatctcc ttcgccaaaa 240gcaggcgccg ccaccgggcg catcgtggcg gtcattggcg cagtggtgga cgtccagttt 300gatgagggac taccaccaat tctaaatgcc ctggaagtgc aaggcaggga gaccagactg 360gttttggagg tggcccagca tttgggtgag agcacagtaa ggactattgc tatggatggt 420acagaaggct tggttagagg ccagaaagta ctggattctg gtgcaccaat caaaattcct 480gttggtcctg agactttggg cagaatcatg aatgtcattg gagaacctat tgatgaaaga 540ggtcccatca aaaccaaaca atttgctccc attcatgctg aggctccaga gttcatggaa 600atgagtgttg agcaggaaat tctggtgact ggtatcaagg ttgtcgatct gctagctccc 660tatgccaagg gtggcaaaat tgggcttttt ggtggtgctg gagttggcaa gactgtactg 720atcatggagt taatcaacaa tgtcgccaaa gcccatggtg gttactctgt gtttgctggt 780gttggtgaga ggacccgtga aggcaatgat ttataccatg aaatgattga atctggtgtt 840atcaacttaa aagatgccac ctctaaggta gcgctggtat atggtcaaat gaatcaacca 900cctggtgctc gtgcccgggt agctctgact gggctgactg tggctgaata cttcagagac 960caagaaggtc aagatgtact gctatttatt gataacatct ttcgcttcac ccaggctggt 1020tcagaggtgt ctgcattatt gggccgaatc ccttctgctg tgggctatca gcctaccctg 1080gccactgaca tgggcactat gcaggaaaga attaccacta ccaagaaggg atctatcacc 1140tctgtacagg ctatctatgt gcctgctgat gacttgactg accctgcccc tgctactacg 1200tttgcccatt tggatgctac cactgtactg tcgcgtgcca ttgctgagct gggcatctat 1260ccagctgtgg atcctctaga ctccacctct cgtatcatgg atcccaacat tgttggcagt 1320gagcattacg atgttgcccg tggggtgcaa aagatcctgc aggactacaa atccctccag 1380gatatcattg ccatcctggg tatggatgaa ctttctgagg aagacaagtt gaccgtgtcc 1440cgtgcacgga aaatacagcg tttcttgtct cagccattcc aggttgctga ggtcttcaca 1500ggtcatatgg ggaagctggt acccctgaag gagaccatca aaggattcca gcagattttg 1560gcaggtgaat atgaccatct cccagaacag gccttctata tggtgggacc cattgaagaa 1620gctgtggcaa aagctgataa gctggctgaa gagcattcat cgtgaggggt ctttgtcctc 1680tgtacttgtc tctctccttg cccctaaccc aaaaagcttc atttttctat ataggctgca 1740caagagcctt gattgaagat atattctttc tgaacagtat ttaaggtttc caataaaatc 1800ggaattc 1807 5 4992 DNA Homo sapiens any n or Xaa = unknown 5 ccgcggtgagccgcgaggaa gagaggcgag cgagagtgga ggaggaggcg gcggctgcgg 60 gacggtccccaggaatgtcg ctgccccccc cccccctgcc gttgaggagg agacggagga 120 gaccgacgttgttagggaag atgatcccta tgatctgccg ctgtttctgc acagaaatga 180 gggaaatacaaagaaccaaa tacagttcta aatttgggat ctgtattttg agatgatttt 240 attttcagaatgagaagcat atctggttac ctttatgaat gtagagacat gagaagagag 300 ttatgatggcaaaaaacaaa gagcctcgtc ccccatccta taccatcagt atagttggac 360 tctctgggactgaaaaagac aaaggtaact gtggagttgg aaagtcttgt ttgtgcaata 420 gatttgtacgctcaaaagca gatgaatatt atccagagca tacttctgtg cttagcacca 480 ttgactttggaggacgagta gtaaacaatg atcacttttt gtactggggt gacataatac 540 aaaatagtgaagatggagta gaatgcaaaa ttcatgtcat tgaacaaaca gagttcattg 600 atgaccagactttcttgcct catcggagta cgaatttgca accatatata aaacgtgcag 660 ctgcatctaaattgcagtca gcagaaaaac taatgtacat ttgcactgat cagctaggct 720 tagaacaagactttgaacag aagcaaatgc ctgaagggaa gctcaacgta gatggatttt 780 tattatgcattgatgtaagt caaggatgca ataggaagtt tgatgatcaa cttaaatttg 840 tgaataacctttttgtccag ttatcaaaat caaaaaaacc tgtaataata gcagcaacta 900 aatgtgatgaatgcgtgggt cattatctta gagaagttca ggcatttgct tcaaataaaa 960 agaaccttcttgtagtggaa acactcagcg caataaaagt caacattgaa acatgtttta 1020 ctgcactggtacaaatgttg gataaaactc gtagcaagcc taaaattatt ccctatttgg 1080 atgcttataaaacacagaga caacttgttg tcacagcaac agataagttt gaaaaacttg 1140 tgcagactgtgagagattat catgcaactt ggaaaactgt tagtaataaa ttaaaaaatc 1200 atcctgattatgaagaatac atcaacttag agggaacaag aaaggccaga aatacattct 1260 caaaacatatagaacaactt aaacaggaac atataagaaa aaggagagaa gagtatataa 1320 atactttaccaagagctttt aacactcttt tgccaaatct agaagagatt gaacatttga 1380 attggtcagaagctttgaag ttaatggaaa agagagcaga tttccagtta tgttttgtgg 1440 tgctagaaaaaactccttgg gatgaaactg accatataga caaaattaat gataggcgga 1500 ttccatttgacctcctgagc actttagaag ctgaaaaagt ctatcagaac catgtacagc 1560 atctgatatccgagaagagg agggtggaaa tgaaggaaaa attcaaaaag actttggaaa 1620 aaattcaattcatttcacca gggcagccat gggaggaagt tatgtgcttt gttatggagg 1680 atgaagcctacaaatatatc actgaggctg atagcaaaga ggtatatggt aggcatcagc 1740 gagaaatagttgaaaaagcc aaagaagagt ttcaagaaat gctttttgag cattctgaac 1800 ttttttatgatttagatctt aatgcaacac ctagttcaga taaaatgagt gaaattcata 1860 cagttctgagtgaagaacct agatataaag ctttacagaa acttgcacct gatagggaat 1920 cccttctacttaagcatata ggatttgttt atcatcccac taaagaaaca tgtcttagtg 1980 gccaaaattgtacagacatt aaagtggagc agttacttgc tagtagtctt ttacagttgg 2040 atcatggccgcttaagatta tatcacgata gtaccaatat agataaagtt aaccttttta 2100 ttttagggaaggatggcctt gcccaagaac tagcaaatga gataaggaca caatccactg 2160 atgatgagtatgccttagat ggaaaaattt atgaacttga tcttcggccg gttgatgcca 2220 aatcgccttactttttgagt cagttatgga ctgccgcctt taaaccacat gggtgcttct 2280 gtgtatttaattccattgag tcattgagtt ttattgggga atttattggg aaaataagaa 2340 ctgaagcttctcagatcaga aaagataaat acatggctaa tcttccattt acattaattc 2400 tggctaatcagagagattcc attagtaaga atctaccaat tctcaggcac caagggcagc 2460 agttggcaaacaagttgcaa tgtccttttg tagatgtacc tgctggtaca tatcctcgta 2520 aatttaatgaaacccaaata aagcaagctc tcagaggagt attggaatca gttaaacaca 2580 atttggatgtggtgagccca attcctgcca ataaggactt atcagaagct gacttgagaa 2640 ttgtcatgtgcgccatgtgt ggagatccat ttagtgtgga tcttattctt tcacccttcc 2700 ttgattctcattcttgcagt gctgctcaag ctggacagaa taattcccta atgcttgata 2760 aaatcattggtgaaaaaagg aggcgaatac agatcacaat attatcatac cactcttcaa 2820 ttggagtaagaaaagatgaa ctagttcatg ggtatatatt agtttactct gcaaaacgga 2880 aagcttcgatgggaatgctt cgagcatttc tatcagaagt tcaagacacc attcctgtac 2940 agctggtggcagttactgac agccaagcag atttttttga aaatgaggct atcaaagagt 3000 taatgactgaaggagaacac attgcaactg agatcactgc taaatttaca gcactgtatt 3060 ctttatctcagtatcatcgg caaactgagg tctttactct gttttttagt gatgttctag 3120 agaaaaaaaatatgatagaa aattcttatt tgtctgataa tacaagggaa tcaacccatc 3180 aaagtgaagatgtttttcta ccatctccca gagactgttt tccctataat aactaccctg 3240 attcagatgatgacacagaa gcaccacctc cttatagtcc aattggggat gatgtacagt 3300 tgcttccaacacctagtgac cgttccagat atagattaga tttggaagga aatgagtatc 3360 ctattcatagtaccccaaac tgtcatgacc atgaacgcaa ccataaagtg cctccaccta 3420 ttaaacctaaaccagttgta cctaagacaa atgtgaaagc gctcgttcca aaccttttaa 3480 gggcaattgaagctggtatt ggtaaaaatc caagaaagca gacttcccgg gtgcctttcg 3540 gtcctgaagatatggatcct tcagataact atgcggaacc cattgataca attttcaaac 3600 agaagggctattctgatgag atttatgttg tcccagatga tagtcaaaat cgtattaaaa 3660 ttcgaaactcatttgtaaat aacacccaag gagatgaaga aaatgggttt tctgatagac 3720 ctcaaaaagtcatggggaac ggaggccttc aaaatacaaa tataaatcta aaaccttgtt 3780 tagtaaagccaagtcatact atagaagaac acattcagat gccagtgatg atgaggcttt 3840 caccacttctaaaaccaaaa agaaaaggaa gacatcgtgg aagtgaagaa gatccacttc 3900 tttctcctgttgaaacttgg aaaggtggta ttgataatcc tgcaatcact tctgaccagg 3960 agttagatgataagaagatg aagaagaaaa cccacaaagt gaaagaagat aaaaaaaaga 4020 aaactaagaacttcaatcca ccaacacgta gaaattggga aagtaattac tttgggatgc 4080 ccctccaggatctggttaca gctgagaagc ccataccact atttgttgag aaatgtgtgg 4140 aatttattgaagatacaggg ttatgtaccg agagactcta ccgtgtcagc gggaataaaa 4200 ctgaccaagaaaatattcaa aagcagtttg ttcaagatca taatatcaat ctagtgtcaa 4260 tggaagtaacagtaaatgct gtagctggag cccttaaagc tttctttgca gatctgccag 4320 atcctttaattccatattct cttcatccag aactattgga agcagcaaaa atcccggata 4380 aaacagaacgtcttcatgcc ttgaaagaaa ttgttaagaa atttcatcct gtaaactatg 4440 atgtattcagatacgtgata acacatctaa acagggttag tcagcaacat aaaatcaacc 4500 taatgacagcagacaactta tccatctgtt ttggccaacc cttgatgaga cctgatttga 4560 aatcgatggagtttctgtct actactaaga ttcatcaatc tgttgttgaa acattcattc 4620 agcagtgtcagtttttcttt tacaatggag aaattgtaga aacgacaaac attgtggctc 4680 ctccaccaccttcaaaccca ggacagttgg tggaaccaat ggtgccactt cagttgccgc 4740 caccattgcaacctcagctg atacaaccac aattacaaac ggatcctctt ggtattatat 4800 gagtaggaagtgattgcaaa caggctggat ttggacaaaa agcaaatcta gacatgcatg 4860 tttcagggttcagtagtata cttcatgttt catacagata attcacattc aaaattacat 4920 tttctctttgaactagatgg tattccttat tcacttacat tacaaatcta agaccatgtg 4980 ataagcatgact 4992 6 708 DNA Homo sapiens any n or Xaa = unknown 6 tatggcacatgcagcgcaag taggtctaca agacgctact tcccctatca tagaagagct 60 tatcacctttcatgatcacg ccctcataat cattttcctt atctgcttcc tagtcctgta 120 tgcccttttcctaacactca caacaaaact aactaatact aacatctcag acgctcagga 180 aatagaaaccgtctgaacta tcctgcccgc catcatccta gtcctcatcg ccctcccatc 240 cctacgcatcctttacataa cagacgaggt caacgatccc tcccttacca tcaaatcaat 300 tggccaccaatggtactgaa cctacgagta caccgactac ggcggactaa tcttcaactc 360 ctacatacttcccccattat tcctagaacc aggcgacctg cgactccttg acgttgacaa 420 tcgagtagtactcccgattg aagcccccat tcgtataata attacatcac aagacgtctt 480 gcactcatgagctgtcccca cattaggctt aaaaacagat gcaattcccg gacgtctaaa 540 ccaaaccactttcaccgcta cacgaccggg ggtatactac ggtcaatgct ctgaaatctg 600 tggagcaaaccacagtttca tgcccatcgt cctagaatta attcccctaa aaatctttga 660 aatagggcccgtatttaccc tatagcaccc cctctacccc ctctagag 708 7 1140 DNA Homo sapiensany n or Xaa = unknown 7 atgaccccaa tacgcaaaat taacccccta ataaaattaattaaccactc attcatcgac 60 ctccccaccc catccaacat ctccgcatga tgaaacttcggctcactcct tggcgcctgc 120 ctgatcctcc aaatcaccac aggactattc ctagccatgcactactcacc agacgcctca 180 accgcctttt catcaatcgc ccacatcact cgagacgtaaattatggctg aatcatccgc 240 taccttcacg ccaatggcgc ctcaatattc tttatctgcctcttcctaca catcgggcga 300 ggcctatatt acggatcatt tctctactca gaaacctgaaacatcggcat tatcctcctg 360 cttgcaacta tagcaacagc cttcataggt tatgtcctcccgtgaggcca aatatcattc 420 tgaggggcca cagtaattac aaacttacta tccgccatcccatacattgg gacagaccta 480 gttcaatgaa tctgaggagg ctactcagta gacagtcccaccctcacacg attctttacc 540 tttcacttca tcttgccctt cattattgca accctagcagcactccacct cctattcttg 600 cacgaaacgg gatcaaacaa ccccctagga atcacctcccattccgataa aatcaccttc 660 cacccttact acacaatcaa agacaccctc ggcttacttctcttccttct ctccttaatg 720 acattaacac tattctcacc agacctccta ggcgacccagacaattatac cctagccaac 780 cccttaaaca cccctcccca catcaagccc gaatgatatttcctattcgc ctacacaatt 840 ctccgatccg tccctaacaa actaggaggc gtccttgccctattactatc catcctcatc 900 ctagcaataa tccccatcct ccatatatcc aaacaacaaagcataatatt tcgcccacta 960 agccaatcac tttattgact cctagccgca gacctcctcattctaacctg aatcggagga 1020 caaccagtaa gctacccttt taccatcatt ggacaagtagcatccgtact atacttcaca 1080 acaatcctaa tcctaatacc aactatctcc ctaattgaaaacaaaatact caaatgggcc 1140 8 5629 DNA Homo sapiens any n or Xaa =unknown 8 gcgcgaccgt cccgggggtg gggccgggcg cagcggcgag aggaggcgaaggtggctgcg 60 gtagcagcag cgcggcagcc tcggacccag cccggagcgc agggcggccgctgcaggtcc 120 ccgctcccct ccccgtgcgt ccgcccatgg ccgccgccgg gcagctgtgcttgctctacc 180 tgtcggcggg gctcctgtcc cggctcggcg cagccttcaa cttggacactcgggaggaca 240 acgtgatccg gaaatatgga gaccccggga gcctcttcgg cttctcgctggccatgcact 300 ggcaactgca gcccgaggac aagcggctgt tgctcgtggg ggccccgcgcggagaagcgc 360 ttccactgca gagagccaac agaacgggag ggctgtacag ctgcgacatcaccgcccggg 420 ggccatgcac gcggatcgag tttgataacg atgctgaccc cacgtcagaaagcaaggaag 480 atcagtggat gggggtcacc gtccagagcc aaggtccagg gggcaaggtcgtgacatgtg 540 ctcaccgata tgaaaaaagg cagcatgtta atacgaagca ggaatcccgagacatctttg 600 ggcggtgtta tgtcctgagt cagaatctca ggattgaaga cgatatggatgggggagatt 660 ggagcttttg tgatgggcga ttgagaggcc atgagaaatt tggctcttgccagcaaggtg 720 tagcagctac ttttactaaa gactttcatt acattgtatt tggagccccgggtacttata 780 actggaaagg gattgttcgt gtagagcaaa agaataacac tttttttgacatgaacatct 840 ttgaagatgg gccttatgaa gttggtggag agactgagca tgatgaaagtctcgttcctg 900 ttcctgctaa cagttactta ggtttttctt tggactcagg gaaaggtattgtttctaaag 960 atgagatcac ttttgtatct ggtgctccca gagccaatca cagtggagccgtggttttgc 1020 tgaagagaga catgaagtct gcacatctcc tccctgagca catattcgatggagaaggtc 1080 tggcctcttc atttggctat gatgtggcgg tggtggacct caacaaggatgggtggcaag 1140 atatagttat tggagcccca cagtattttg atagagatgg agaagttggaggtgcagtgt 1200 atgtctacat gaaccagcaa ggcagatgga ataatgtgaa gccaattcgtcttaatggaa 1260 ccaaagattc tatgtttggc attgcagtaa aaaatattgg agatattaatcaagatggct 1320 acccagatat tgcagttgga gctccgtatg atgacttggg aaaggtttttatctatcatg 1380 gatctgcaaa tggaataaat accaaaccaa cacaggttct caagggtatatcaccttatt 1440 ttggatattc aattgctgga aacatggacc ttgatcgaaa ttcctaccctgatgttgctg 1500 ttggttccct ctcagattca gtaactattt tcagatcccg gcctgtgattaatattcaga 1560 aaaccatcac agtaactcct aacagaattg acctccgcca gaaaacagcgtgtggggcgc 1620 ctagtgggat atgcctccag gttaaatcct gttttgaata tactgctaaccccgctggtt 1680 ataatccttc aatatcaatt gtgggcacac ttgaagctga aaaagaaagaagaaaatctg 1740 ggctatcctc aagagttcag tttcgaaacc aaggttctga gcccaaatatactcaagaac 1800 taactctgaa gaggcagaaa cagaaagtgt gcatggagga aaccctgtggctacaggata 1860 atatcagaga taaactgcgt cccattccca taactgcctc agtggagatccaagagccaa 1920 gctctcgtag gcgagtgaat tcacttccag aagttcttcc aattctgaattcagatgaac 1980 ccaagacagc tcatattgat gttcacttct taaaagaggg atgtggagacgacaatgtat 2040 gtaacagcaa ccttaaacta gaatataaat tttgcacccg agaaggaaatcaagacaaat 2100 tttcttattt accaattcaa aaaggtgtac cagaactagt tctaaaagatcagaaggata 2160 ttgctttaga aataacagtg acaaacagcc cttccaaccc aaggaatcccacaaaagatg 2220 gcgatgacgc ccatgaggct aaactgattg caacgtttcc agacactttaacctattctg 2280 catatagaga actgagggct ttccctgaga aacagttgag ttgtgttgccaaccagaatg 2340 gctcgcaagc tgactgtgag ctcggaaatc cttttaaaag aaattcaaatgtcacttttt 2400 atttggtttt aagtacaact gaagtcacct ttgacacccc atatctggatattaatctga 2460 agttagaaac aacaagcaat caagataatt tggctccaat tacagctaaagcaaaagtgg 2520 ttattgaact gcttttatcg gtctcgggag ttgctaaacc ttcccaggtgtattttggag 2580 gtacagttgt tggcgagcaa gctatgaaat ctgaagatga agtgggaagtttaatagagt 2640 atgaattcag ggtaataaac ttaggtaaac ctcttacaaa cctcggcacagcaaccttga 2700 acattcagtg gccaaaagaa attagcaatg ggaaatggtt gctttatttggtgaaagtag 2760 aatccaaagg attggaaaag gtaacttgtg agccacaaaa ggagataaactccctgaacc 2820 taacggagtc tcacaactca agaaagaaac gggaaattac tgaaaaacagatagatgata 2880 acagaaaatt ttctttattt gctgaaagaa aataccagac tcttaactgtagcgtgaacg 2940 tgaactgtgt gaacatcaga tgcccgctgc gggggctgga cagcaaggcgtctcttattt 3000 tgcgctcgag gttatggaac agcacatttc tagaggaata ttccaaactgaactacttgg 3060 acattctcat gcgagccttc attgatgtga ctgctgctgc cgaaaatatcaggctgccaa 3120 atgcaggcac tcaggttcga gtgactgtgt ttccctcaaa gactgtagctcagtattcgg 3180 gagtaccttg gtggatcatc ctagtggcta ttctcgctgg gatcttgatgcttgctttat 3240 tagtgtttat actatggaag tgtggtttct tcaagagaaa taagaaagatcattatgatg 3300 ccacatatca caaggctgag atccatgctc agccatctga taaagagaggcttacttctg 3360 atgcatagta ttgatctact tctgtaattg tgtggattct ttaaacgctctaggtacgat 3420 gacagtgttc cccgatacca tgctgtaagg atccggaaag aagagcgagagatcaaagat 3480 gaaaagtata ttgataacct tgaaaaaaaa cagtggatca caaagtggaacagaaatgaa 3540 agctactcat agcgggggcc taaaaaaaaa aaagcttcac agtacccaaactgctttttc 3600 caactcagaa attcaatttg gatttaaaag cctgctcaat ccctgaggactgatttcaga 3660 gtgactacac acagtacgaa cctacagttt taactgtgga tattgttacgtagcctaagg 3720 ctcctgtttt gcacagccaa atttaaaact gttggaatgg atttttctttaactgccgta 3780 atttaacttt ctgggttgcc tttgtttttg gcgtggctga cttacatcatgtgttgggga 3840 agggcctgcc cagttgcact caggtgacat cctccagata gtgtagctgaggaggcacct 3900 acactcacct gcactaacag agtggccgtc ctaacctcgg gcctgctgcgcagacgtcca 3960 tcacgttagc tgtcccacat cacaagacta tgccattggg gtagttgtgtttcaacggaa 4020 agtgctgtct taaactaaat gtgcaataga aggtgatgtt gccatcctaccgtcttttcc 4080 tgtttcctag ctgtgtgaat acctgctcac gtcaaatgca tacaagtttcattctccctt 4140 tcactaaaaa cacacaggtg caacagactt gaatgctagt tatacttatttgtatatggt 4200 atttattttt tcttttcttt acaaaccatt ttgttattga ctaacaggccaaagagtctc 4260 cagtttaccc ttcaggttgg tttaatcaat cagaattaga attagagcatgggagggtca 4320 tcactatgac ctaaattatt tactgcaaaa agaaaatctt tataaatgtaccagagagag 4380 ttgttttaat aacttatcta taaactataa cctctccttc atgacagcctccaccccaca 4440 acccaaaagg tttaagaaat agaattataa ctgtaaagat gtttatttcaggcattggat 4500 attttttact ttagaagcct gcataatgtt tctggattta catactgtaacattcaggaa 4560 ttcttggaga agatgggttt attcactgaa ctctagtgcg gtttactcactgctgcaaat 4620 actgtatatt caggacttga aagaaatggt gaatgcctat ggaactagtggatccaaact 4680 gatccagtat aagactactg aatctgctac caaaacagtt aatcagtgagtcgagtgttc 4740 tattttttgt tttgtttcct cccctatctg tattcccaaa aattactttggggctaattt 4800 aacaagaact ttaaattgtg ttttaattgt aaaaatggca gggggtggaattattactct 4860 atacattcaa cagagactga atagatatga aagctgattt tttttaattaccatgcttca 4920 caatgttaag ttatatgggg agcaacagca aacaggtgct aatttgttttggatatagta 4980 taagcagtgt ctgtgttttg aaagaataga acacagtttg tagtgccactgttgttttgg 5040 ggggggcttt ttttcttttt ccggaaaatc cttaaacctt aagatactaaggacgttgtt 5100 ttggttgtac ttggaattct tagtcacaaa atatattttg tttacaaaaatttctgtaaa 5160 acaggttata acagtgttta aagtctcagt ttcttgcttg gggaacttgtgtccctaatg 5220 tgttagattg ctagattgct aaggagctga tacttgacag ttttttagacctgtgttact 5280 aaaaaaaaga tgaatgtcgg aaaagggtgt tgggagggtg gtcaacaaagaaacaaagat 5340 gttatggtgt ttagacttat ggttgttaaa aatgtcatct caagtcaagtcactggtctg 5400 tttgcatttg atacattttt gtactaacta gcattgtaaa attatttcatgattagaaat 5460 tacctgtgga tatttgtata aaagtgtgaa ataaattttt tataaaagtgttcattgttt 5520 cgtaacacag cattgtatat gtgaagcaaa ctctaaaatt ataaatgacaacctgaatta 5580 tctatttcat caaaaaaaaa aaaaaaaaaa actttatggg cacaactgg5629 9 580 DNA Homo sapiens any n or Xaa = unknown 9 ccatccaatgaggccacctc tttctaaact cagactcttc atttagggag gtgagttcca 60 ttaaggaacttgagattttc agataaatgg aaaatactag ataaagaggt atctcataga 120 tagcaaaggtaaactctcat acaatcattg agctaggaca ttaatggttc agtggttccc 180 aattctagatatacattaaa ataaattgaa aagcctttta aaaatacatg attactggac 240 ctactgaattatatcctttg gggagcccaa gaacttatta aattctctgg gctattttta 300 tgatttctctgagctgttac tgggaactac tgattgaatc catyttttat agtaatgttt 360 ccaacagaaggctgtttscc tttgcttaac attatttcca gtgaagtatt attttccatt 420 ctggagacagttcaaaagtt tttttaagta acagctttat tgagacaatt tatatsccgt 480 acaattcacctaaagtgtgt aattcagttg tttttagtat gttcacagaa ttgtgcagct 540 tgcatctatcaccacaaatt tagaaccttg tcataatccc 580 10 1552 DNA Homo sapiens any n orXaa = unknown 10 cccaaaccca ctccacctta ctaccagaca accttagcca aaccatttacccaaataaag 60 tataggcgat agaaattgaa acctggcgca atagatatag taccgcaagggaaagatgaa 120 aaattataac caagcataat atagcaagga ctaaccccta taccttctgcataatgaatt 180 aactagaaat aactttgcaa ggagagccaa agctaagacc cccgaaaccagacgagctac 240 ctaagaacag ctaaaagagc acacccgtct atgtagcaaa atagtgggaagatttatagg 300 tagaggcgac aaacctaccg agcctggtga tagctggttg tccaagatagaatcttagtt 360 caactttaaa tttgcccaca gaaccctcta aatccccttg taaatttaactgttagtcca 420 aagaggaaca gctctttgga cactaggaaa aaaccttgta gagagagtaaaaaatttaac 480 acccatagta ggcctaaaag cagccaccaa ttaagaaagc gttcaagctcaacacccact 540 acctaaaaaa tcccaaacat ataactgaac tcctcacacc caattggaccaatctatcac 600 cctatagaag aactaatgtt agtataagta acatgaaaac attctcctccgcataagcct 660 gcgtcagatt aaaacactga actgacaatt aacagcccaa tatctacaatcaaccaacaa 720 gtcattatta ccctcactgt caacccaaca caggcatgct cataaggaaaggttaaaaaa 780 agtaaaagga actcggcaaa tcttaccccg cctgtttacc aaaaacatcacctctagcat 840 caccagtatt agaggcaccg cctgcccagt gacacatgtt taacggccgcggtaccctaa 900 ccgtgcaaag gtagcataat cacttgttcc ttaattaggg acccgtatgaatggctccac 960 gagggttcag ctgtctctta cttttaacca gtgaaattga cctgcccgtgaagaggcggg 1020 catgacacag caagacgaga agaccctatg gagctttaat ttattaatgcaaacagtacc 1080 taacaaacct acaggtccta aactaccaaa cctgcattaa aaatttcggttggggcgacc 1140 tcggagcaga acccaacctc cgagcagtac atgctaagac ttcaccagtcaaagcgaact 1200 actatactca attgatccaa taacttgacc aacggaacaa gttaccctagggataacagc 1260 gcaatcctat tctagagtcc atatcaacaa tagggtttac gacctcgatgttggatcagg 1320 acatcccgat ggtgcagccg ctattaaagg ttcgtttgtt caacgattaaagtcctacgt 1380 gatctgagtt cagaccggag taatccaggt cggtttctat ctacttcaaattcctccctg 1440 tacgaaagga caagagaaat aaggcctact tcacaaagcg ccttcccccgtaaatgatat 1500 catctcaact tagtattata cccacaccca cccaagaaca gggtttgttaag 1552 11 2116 DNA Homo sapiens any n or Xaa = unknown 11 gggtggcagaatattagtct agctatctcc cattgctctc acgcgccatc tactggattt 60 catcccaaactacaacacga aaaactgcta attttcctgc ctgccaggcc gaggactgga 120 attcaacagactgtttagag cctttgccct ctgaaaactt ccagaaatga agccaactga 180 ctatattcagtttacaccag agttaaagga acgccaaccc tcccagatga gaaagaatca 240 gtgcaagaactgtagcaatt taaaaaacca gagcgtcccc ttacctccaa atgagcccac 300 tagctccacagcaattgttc ttaaccaatc tgaaatgatg agcatggaat tcagaatctg 360 aatggcaatgaagcttatag atatccagga gaaagttgaa atgcaatcca aggaaaccaa 420 gcaatccagtgaaatggttt aagagctgaa agataaaata ncaattttac aaaagaccca 480 aactgagcttattgagttca aaaaagaatt tcataataca atcagaagta ttaatagcag 540 aataggccaagctgaggaaa gaatctcaga gcttgacccc tggttctttg aatcaactta 600 gacaaaaataaagaaaaaag agttttaaga aatgaacaca atctcccaga aatatgagat 660 tatgtwaagagacaaaatct atgactcatt gccatccctg agagagaagg agagagaata 720 agcaacttggaaaatatatt tggggacata gcccacaaaa atttccctaa tctctctaga 780 gaggttgacatgtaaattca agaaatacag aagaccttgg ccagataata tacaagatga 840 ccatccccaaggcacatagt catcagattc accatggtca atgcaaaaga aaaaaatctt 900 aaagacagctagggagaagg gtcaagtcac atgcagaagg actctcatta ggctggcagt 960 ggacctctcagcagaaacct gacaagccag aagagatgga gggagagggg tctatttttg 1020 tcatccttaaagaaaaaaaa ttccaaccaa gagtctcata cactgccaaa ctaagcttcc 1080 taagtgaaggagaaataaaa accttctcag acaagcaaat gctgaaggaa ttcaactaga 1140 ccagcctaacaagaggtcct aagggagtgc tgaatatgga ctcaaaagaa taacacctgc 1200 taccacaaacactcacttaa gcacacagcc caacgacact ataggcaatt acacagtaag 1260 tctacataacaacacaatga caggatcaac atctcacaca tcaatactaa ccccgagtgt 1320 aaaggggctaaatgccccac ttaaaagaca tagagtgtca agcttgataa aaagacaaga 1380 tccaatcatccactattttc aagagctcta tgttatgtgt aatgacaccc acagactcaa 1440 agacttggagaaagatttat catgcaaaat cagaaaacaa aaaagagcag gagtcactag 1500 ttttatatcagacaaaacag actttaaacc cttaataatt aagaaagaca aagaagggta 1560 tttcctggaccacagaaggc ttattggaaa aaaggacata atgacaaagg gtacaatcca 1620 acaagaagttttaactattc taaatatata cacacccaac attggagcac ccagatttat 1680 aaaacaagtacttctcgatc tacaagaaga cttagacagc cacacaataa tagtgggaga 1740 ctttcacatcctacttacag atcattgaga cagaaaacta ataaaagaac tctggactta 1800 aacttgttacttgaccaatt ggacctaata gatatccaca gaaaacttca cccaacaaag 1860 acagaatatacattcttctt atctgcacat ggaacacatt ccaagatcaa tcacatgcta 1920 ggtaagaaagcaagtctcaa taaattaaaa aaaattgaaa tcatacgaac cttaatatca 1980 gaccacaatgtaattaaaaa taaatcaata tcaagaagat ctcatacata aatacatgaa 2040 aattaaacaacttactcctg aataactctt gtgtgaacat caaaattcag gaagaaataa 2100 aaaattatttgaaatt 2116 12 173 DNA Homo sapiens any n or Xaa = unknown 12 gcgatccacaaatgggaggt gacggtccat cagggaagct gggttcgcgg ctccacggct 60 gggggctgccgcaatttcct ggataccttt tggaccaatc cacaaataaa attgtctctg 120 actgagaaagatgaggggca ggaggagtgt agtttccttg tagccctgat gca 173 13 655 DNA Homosapiens any n or Xaa = unknown 13 ctgatccatg ggccagcagc atcaatattacctgggagct tacagaaatg cagaatttca 60 ggcccactgc agatctaccg aatcaaaatcttcctttagc aaaatttctc aaacgattag 120 cactggccta catccatttt atccttccttagctattagg gatgtgaggt ccgagggctt 180 caaaaggtcc ccggaatagc ttgttccttcatccactgtg tcctattcat tcttcagcta 240 actccagcaa tgagctgaaa ctcattcatcacccttgctg agttttcttc tcaatcctta 300 ttcctaattc tggttctaga tgagccctacctacccagtg gttgtatttt tgtagccagt 360 gtgggacaca ggagattggc agaccaacacagctagcctc tctctagccc tccctccacc 420 tctaagtcac taacaatcca tgtttgttcagtttgttgac atgtggcatg ttcatttgtt 480 cacaacttaa tcacggggga catttcagaaaaatgtgtac taagttaaaa ccatgtttag 540 tctcctacaa cttgtacatt ttcattttctcttatcagta gattgtcctt gttgacatag 600 ctcatgcatg aggacacata gcagtacacacacattgaat gaattgttag tcatg 655 14 2619 DNA Homo sapiens any n or Xaa =unknown 14 gactcctagg ggcttgcaga cctagtggga gagaaagaac atcgcagcagccaggcagaa 60 ccaggacagg tgaggtgcag gctggctttc ctctcgcagc gcggtgtggagtcctgtcct 120 gcctcagggc ttttcggagc ctggatcctc aaggaacaag tagacctggccgcggggagt 180 ggggagggaa ggggtgtcta ttgggcaaca gggcggcaaa gccctgaataaaggggcgca 240 gggcaggcgc aagtgcagag ccttcgtttg ccaagtcgcc tccagaccgcagacatgaaa 300 cttgtcttcc tcgtcctgct gttcctcggg gccctcggac tgtgtctggctggccgtagg 360 agaaggagtg ttcagtggtg cgccgtatcc caacccgagg ccacaaaatgcttccaatgg 420 caaaggaata tgagaaaagt gcgtggccct cctgtcagct gcataaagagagactccccc 480 atccagtgta tccaggccat tgcggaaaac agggccgatg ctgtgacccttgatggtggt 540 ttcatatacg aggcaggcct ggccccctac aaactgcgac ctgtagcggcggaagtctac 600 gggaccgaaa gacagccacg aactcactat tatgccgtgg ctgtggtgaagaagggcggc 660 agctttcagc tgaacgaact gcaaggtctg aagtcctgcc acacaggccttcgcaggacc 720 gctggatgga atgtccctac agggacactt cgtccattct tgaattggacgggtccacct 780 gagcccattg aggcagctgt ggccaggttc ttctcagcca gctgtgttcccggtgcagat 840 aaaggacagt tccccaacct gtgtcgcctg tgtgcgggga caggggaaaacaaatgtgcc 900 ttctcctccc aggaaccgta cttcagctac tctggtgcct tcaagtgtctgagagacggg 960 gctggagacg tggcttttat cagagagagc acagtgtttg aggacctgtcagacgaggct 1020 gaaagggacg agtatgagtt actctgccca gacaacactc ggaagccagtggacaagttc 1080 aaagactgcc atctggcccg ggtcccttct catgccgttg tggcacgaagtgtgaatggc 1140 aaggaggatg ccatctggaa tcttctccgc caggcacagg aaaagtttggaaaggacaag 1200 tcaccgaaat tccagctctt tggctcccct agtgggcaga aagatctgctgttcaaggac 1260 tctgccattg ggttttcgag ggtgcccccg aggatagatt ctgggctgtaccttggctcc 1320 ggctacttca ctgccatcca gaacttgagg aaaagtgagg aggaagtggctgcccggcgt 1380 gcgcgggtcg tgtggtgtgc ggtgggcgag caggagctgc gcaagtgtaaccagtggagt 1440 ggcttgagcg aaggcagcgt gacctgctcc tcggcctcca ccacagaggactgcatcgcc 1500 ctggtgctga aaggagaagc tgatgccatg agtttggatg gaggatatgtgtacactgca 1560 tgcaaatgtg gtttggtgcc tgtcctggca gagaactaca aatcccaacaaagcagtgac 1620 cctgatccta actgtgtgga tagacctgtg gaaggatatc ttgctgtggcggtggttagg 1680 agatcagaca ctagccttac ctggaactct gtgaaaggca agaagtcctgccacaccgcc 1740 gtggacagga ctgcaggctg gaatatcccc atgggcctgc tcttcaaccagacgggctcc 1800 tgcaaatttg atgaatattt cagtcaaagc tgtgcccctg ggtctgacccgagatctaat 1860 ctctgtgctc tgtgtattgg cgacgagcag ggtgagaata agtgcgtgcccaacagcaac 1920 gagagatact acggctacac tggggctttc cggtgcctgg ctgagaatgctggagacgtt 1980 gcatttgtga aagatgtcac tgtcttgcag aacactgatg gaaataacaatgaggcatgg 2040 gctaaggatt tgaagctggc agactttgcg ctgctgtgcc tcgatggcaaacggaagcct 2100 gtgactgagg ctagaagctg ccatcttgcc atggccccga atcatgccgtggtgtctcgg 2160 atggataagg tggaacgcct gaaacaggtg ctgctccacc aacaggctaaatttgggaga 2220 aatggatctg actgcccgga caagttttgc ttattccagt ctgaaaccaaaaaccttctg 2280 ttcaatgaca acactgagtg tctggccaga ctccatggca aaacaacatatgaaaaatat 2340 ttgggaccac agtatgtcgc aggcattact aatctgaaaa agtgctcaacctcccccctc 2400 ctggaagcct gtgaattcct caggaagtaa aaccgaagaa gatggcccagctccccaaga 2460 aagcctcagc cattcactgc ccccagctct tctccccagg tgtgttggggccttggctcc 2520 cctgctgaag gtggggattg cccatccatc tgcttacaat tccctgctgtcgtcttagca 2580 agaagtaaaa tgagaaattt tgttgatatt caaaaaaaa 2619 15 892DNA Homo sapiens any n or Xaa = unknown 15 tcttgaccgg cacacacagctcgcttcttc actttctttt ccatccactg ccggacccaa 60 gccagccttc cagggagcagccatgcctta cctctaccgg gccccagggc ctcaggcaca 120 cccggttccc aaggacgcccggatcaccca ctcctcaggc cagarctttg arcaaatgaa 180 gcaggartgc ctgcagarargcaccctgtt tgaggatgca gacttcccag ccagcaattc 240 ctccctgttc tacagtgagaggccgcagat cccctttgtg tggaaacgac cargggaaat 300 cgtgaaaaac ccaraattcattcttggagg ggccaccagg actgatatct gccagggaga 360 gctgggagac tgctggctattagccgccat cgcctccctt acgcttaatc aaaaagcact 420 ggccagagtc atcccccaggaccaaagctt tggccctggt tatgccggga tattccattt 480 ccagttctgg cagcacagtgagtggctgga cgtggtgatc gatgaccgcc tgcccacctt 540 cagggaccgc ttggttttcctccactctgc cgaccacaac garttctgga rcgccttgct 600 ggaaaaagcc tacgccaagctaaatgggag ctatgaagct ctgaagggag gcagcgccat 660 cgaggccatg gaagacttcactgggggtgt ggcagagacc ttccaaacta aagaggcccc 720 cgagaacttc tatgagattctagagaaggc tttgaagana ngctccctgc tgggctgctt 780 cattgatacc agaagtgctgcagaatctga ggcccggacg ccgtttggtc ttattaaggg 840 tcatgcctac agtgtaacgggaattgacca ggtaagcttc cgaggccaga ga 892 16 508 DNA Homo sapiens any n orXaa = unknown 16 tggagaatgc gagccgggtg ttccaggctc tcagtacaaa gaacanggagttcattcatn 60 tcaatataaa ngagttcatc cattngacaa tgaacatctg aggctgcnttgtagagatgc 120 agcctgccca gntgaatctg ggnttctgga cctngacctt cagaanttctcttggtgtgg 180 aaccattacg cccagggttc actcccctct catcgtccgg ccttctcccttcatcttgat 240 ctgggaagaa tgaaatgaac tcagctacac tctctgattt tgtgctactcctttgtaaag 300 tcactgcctt aagggggctg atggcgccac ctgtgcctta catccaggttcaggcatcac 360 tagctttccc acactctact ttccttattt ccttccatta agaattactcagagttctaa 420 cgcacagaat cctgacttcc atgtagctcc agtcattgtg atcagacatcctttataaaa 480 catgttttta taaatgtgta tgtggaat 508 17 194 PRT Homosapiens any n or Xaa = unknown 17 Ser Val His Cys Phe Arg Glu Asp LysMet Lys Phe Thr Ile Val Phe 1 5 10 15 Ala Gly Leu Leu Gly Val Phe LeuAla Pro Ala Leu Ala Asn Tyr Asn 20 25 30 Ile Asn Val Asn Asp Asp Asn AsnAsn Ala Gly Ser Gly Gln Gln Ser 35 40 45 Val Ser Val Asn Asn Glu His AsnVal Ala Asn Val Asp Asn Asn Asn 50 55 60 Gly Trp Asp Ser Trp Asn Ser IleTrp Asp Tyr Gly Asn Gly Phe Ala 65 70 75 80 Ala Thr Arg Leu Phe Gln LysLys Thr Cys Ile Val His Lys Met Asn 85 90 95 Lys Glu Val Met Pro Ser IleGln Ser Leu Asp Ala Leu Val Lys Glu 100 105 110 Lys Lys Leu Gln Gly LysGly Pro Gly Gly Pro Pro Pro Lys Gly Leu 115 120 125 Met Tyr Ser Val AsnPro Asn Lys Val Asp Asp Leu Ser Lys Phe Gly 130 135 140 Lys Asn Ile AlaAsn Met Cys Arg Gly Ile Pro Thr Tyr Met Ala Glu 145 150 155 160 Glu MetGln Glu Ala Ser Leu Phe Phe Tyr Ser Gly Thr Cys Tyr Thr 165 170 175 ThrSer Val Leu Trp Ile Val Asp Ile Ser Phe Cys Gly Asp Thr Val 180 185 190Glu Asn 18 51 PRT Homo sapiens any n or Xaa = unknown 18 Met Val Asp AspLys Arg Lys Ser Ala Leu Trp Lys Glu Arg Thr Val 1 5 10 15 Ser Thr ArgVal Lys Ser Met Asn Ala Ser Ile Glu Arg Thr Arg Gly 20 25 30 Asn Ile ProSer Thr Gly Leu His Thr Cys Ile Tyr Ile Leu Glu Asn 35 40 45 Thr Ala Met50 19 63 PRT Homo sapiens any n or Xaa = unknown 19 Met Gly Gln Gln HisGln Tyr Tyr Leu Gly Ala Tyr Arg Asn Ala Glu 1 5 10 15 Phe Gln Ala HisCys Arg Ser Thr Glu Ser Lys Ser Ser Phe Ser Lys 20 25 30 Ile Ser Gln ThrIle Ser Thr Gly Leu His Pro Phe Tyr Pro Ser Leu 35 40 45 Ala Ile Arg AspVal Arg Ser Glu Gly Phe Lys Arg Ser Pro Glu 50 55 60 20 20 DNAArtificial Sequence any n or Xaa = unknown 20 tctttgctgg acttcttgga 2021 20 DNA Artificial Sequence any n or Xaa = unknown 21 ctttgtttgggttgactgag 20 22 20 DNA Artificial Sequence any n or Xaa = unknown 22caccctcatt acatcatcag 20 23 20 DNA Artificial Sequence any n or Xaa =unknown 23 attccttgtg tcttctggta 20 24 21 DNA Artificial Sequence any nor Xaa = unknown 24 cagtcctact tctcctatct c 21 25 21 DNA ArtificialSequence any n or Xaa = unknown 25 atcatagctc agaccatacc t 21 26 21 DNAArtificial Sequence any n or Xaa = unknown 26 gatcctgcag gactacaaat c 2127 20 DNA Artificial Sequence any n or Xaa = unknown 27 gcctatatagaaaaatgaag 20 28 21 DNA Artificial Sequence any n or Xaa = unknown 28cacctagtga ccgttccaga t 21 29 21 DNA Artificial Sequence any n or Xaa =unknown 29 ttcatctcct tgggtgttat t 21 30 21 DNA Artificial Sequence anyn or Xaa = unknown 30 ctcagacgct caggaaatag a 21 31 21 DNA ArtificialSequence any n or Xaa = unknown 31 aatgggggaa gtatgtagga g 21 32 21 DNAArtificial Sequence any n or Xaa = unknown 32 ttacggatca tttctctact c 2133 21 DNA Artificial Sequence any n or Xaa = unknown 33 agggcaagatgaagtgaaag g 21 34 21 DNA Artificial Sequence any n or Xaa = unknown 34tccggaaaga agagcgagag a 21 35 21 DNA Artificial Sequence any n or Xaa =unknown 35 tgaaacacaa ctaccccaat g 21 36 20 DNA Artificial Sequence anyn or Xaa = unknown 36 atagcaaagg taaactctca 20 37 20 DNA ArtificialSequence any n or Xaa = unknown 37 tcaatcagta gttcccagta 20 38 20 DNAArtificial Sequence any n or Xaa = unknown 38 ttaacagccc aatatctaca 2039 20 DNA Artificial Sequence any n or Xaa = unknown 39 gaacaagtgattatgctacc 20 40 20 DNA Artificial Sequence any n or Xaa = unknown 40agaataagca acttggaaaa 20 41 20 DNA Artificial Sequence any n or Xaa =unknown 41 tgaatctgat gactatgtgc 20 42 20 DNA Artificial Sequence any nor Xaa = unknown 42 tcctggatac cttttggacc 20 43 19 DNA ArtificialSequence any n or Xaa = unknown 43 catcagggct acaaggaaa 19 44 21 DNAArtificial Sequence any n or Xaa = unknown 44 cagatctacc gaatcaaaat c 2145 21 DNA Artificial Sequence any n or Xaa = unknown 45 accagaattaggaataagga t 21 46 20 DNA Artificial Sequence any n or Xaa = unknown 46gactccatgg caaaacaaca 20 47 20 DNA Artificial Sequence any n or Xaa =unknown 47 tcttcttcgg ttttacttcc 20 48 20 DNA Artificial Sequence any nor Xaa = unknown 48 aggcaccagg gcgtgatggt 20 49 20 DNA ArtificialSequence any n or Xaa = unknown 49 ggtctcaaac atgatctggg 20 50 10 DNAArtificial Sequence any n or Xaa = unknown 50 cttgattgcc 10 51 10 DNAArtificial Sequence any n or Xaa = unknown 51 aggtgaccgt 10 52 10 DNAArtificial Sequence any n or Xaa = unknown 52 gttgcgatcc 10 53 10 DNAArtificial Sequence any n or Xaa = unknown 53 ctgatccatg 10 54 10 DNAArtificial Sequence any n or Xaa = unknown 54 ctgcttgatg 10 55 10 DNAArtificial Sequence any n or Xaa = unknown 55 gatctgactg 10 56 13 DNAArtificial Sequence any n or Xaa = unknown 56 tttttttttt taa 13 57 13DNA Artificial Sequence any n or Xaa = unknown 57 tttttttttt tac 13 5813 DNA Artificial Sequence any n or Xaa = unknown 58 tttttttttt tag 1359 13 DNA Artificial Sequence any n or Xaa = unknown 59 tttttttttt tca13 60 13 DNA Artificial Sequence any n or Xaa = unknown 60 tttttttttttcc 13 61 13 DNA Artificial Sequence any n or Xaa = unknown 61tttttttttt tcg 13 62 13 DNA Artificial Sequence any n or Xaa = unknown62 tttttttttt tga 13 63 13 DNA Artificial Sequence any n or Xaa =unknown 63 tttttttttt tgc 13 64 13 DNA Artificial Sequence any n or Xaa= unknown 64 tttttttttt tgg 13 65 264 DNA Artificial Sequence any n orXaa = unknown 65 aggcaccagg gcgtgatggt gggcatgggt cagaaggatt cctatgtgggcgacgaggcc 60 cagagcaaga gaggcatcct caccctgaag taccccatcg agcacggcatcgtcaccaac 120 tgggacgaca tggagaaaat ctggcaccac accttctaca atgagctgcgtgtggctccc 180 gaggagcacc ccgtgctgct gaccgaggcc cccctgaacc ccaaggccaaccgcgagaag 240 atgacccaga tcatgtttga gacc 264 66 814 DNA Homo sapiensany n or Xaa = unknown 66 ataacaccta gtttgagtca acctggttaa gtacaaatatgagaaggctt ctcattcagg 60 tccatgcttg cctactcctc tgtccactgc tttcgtgaagacaagatgaa gttcacaatt 120 gtctttgctg gacttcttgg agtctttcta gctcctgcccttgctaacta taatatcaac 180 gtcaatgatg acaacaacaa tgctggaagt gggcagcagtcagtgagtgt caacaatgaa 240 cacaatgtgg ccaatgttga caataacaac ggatgggactcctggaattc catctgggat 300 tatggaaatg gctttgctgc aaccagactc tttcaaaagaagacatgcat tgtgcacaaa 360 atgaacaagg aagtcatgcc ctccattcaa tcccttgatgcactggtcaa ggaaaagaag 420 cttcagggta agggaccagg aggaccacct cccaagggcctgatgtactc agtcaaccca 480 aacaaagtcg atgacctgag caagttcgga aaaaacattgcaaacatgtg tcgtgggatt 540 ccaacataca tggctgagga gatgcaagag gcaagcctgtttttttactc aggaacgtgc 600 tacacgacca gtgtactatg gattgtggac atttccttctgtggagacac ggtggagaac 660 taaacaattt tttaaagcca ctatggattt agtcgtctgaatatgctgtg cagaaaaaat 720 atgggctcca gtggttttta ccatgtcatt ctgaaatttttctctactag ttatgtttga 780 tttctttaag tttcaataaa atcatttagc attg 814 674646 DNA Homo sapiens any n or Xaa = unknown 67 tatgtgccag gtgctctgttgggtgccaag tgaaatgcaa ataaatggga acagtactca 60 gttcagtttg ctttgggaattaattacatg ccatgtgtgt aaattgtgct aaattttagg 120 aatacagaaa tgaattaaacgtctccaggg aacacatagt ctagtgaaga agctgacaag 180 tgaaaagaga ggatggagtaaaggatttct ggatgccaat gaaaaactac tcgattcttg 240 tatactttca tatgtaagaatttcaagtag caaaaagtca tctgggccct tagaatagca 300 tattttgaag ataataagaaggaagtcact aagaaatgct ctcaggatct agaatagaat 360 tggtatagga aagaggaggccaagcggact tacagacagg gagtaaaaac cctgattcat 420 ctgggtaaca tatgccactgcagatattac tgtcattttt atacaaagtt tctaaatgtg 480 gcagagcaac cagagtgaaagaggtcgggc caactgatga tgaacacaac aaaggaaatt 540 tctcagagta ctggaaggtagataaagaag agtttatgtt tattatatat ctactgccca 600 gaaaaaaatt ttaagtactcattcataaag taaataaagg cacataggta tgccattgac 660 acagaatggc ataatatcactgggattgag ccaaccagca cttccaaaag ttgtcagttt 720 tatttaagct aatgtattattattctaata attccaataa tatatttttt aatgctcttt 780 ctctgaaaaa ttttcccttttccagataat gtcggtgctg gaggctgtgc aaaggctggg 840 ctcctgggca tcttgggaatttcaatctgt gcagacattc atgtttagga tgattagccc 900 tcttgtttta tcttttcaaagaaatacatc cttggtttac actcaaaagt caaattaaat 960 tctttcccaa tgccccaactaattttgaga ttcagtcaga aaatataaat gctgtattta 1020 tagatttttt ggtgtntgttgttttttgta agcagcaaag ggaatccaag caatgtcttt 1080 gtcactatat agaataaaaaaaattgccag aattttaaat aaggtgcata atgtgtgaaa 1140 attcccagat aataccactgggtcacatgt ggactagtca gctggggtcg aatttccatt 1200 tcttcgtntg ccctctggaccagcttccca tctaaccatc caaatatatg ggagcaacct 1260 gggtagagaa gaggctcacacggtggtggc cttgacctgg ccaggggagg gacatagcgt 1320 atgcttatca aacaagttgaatgctcaggt gaaggctttt agggccattc atatgagtta 1380 aaatgtcctt taactcaccaaagcagtaga ctcaacctga ataaacttta taataatatg 1440 tgttgccctg gagtgagaagggagaaaggg agagaggaag gagcacctaa catccaggaa 1500 aagatgcacc atactgaagatcataacagg agtgaaagac tagaaatgcc aagtcaatac 1560 atagcagaaa agcaacttccaatatttcaa ataaattgca cattgtgtac aaatctcaga 1620 tcgtgaagct gggtcacacgtgaacgttcg gctgaatgca aattcagagc aaagaggaat 1680 tactttaata acaatttattctcttgccgt agacctctgg gatcctagct gcagaggacc 1740 cccggcctcc gcgtttgagctgacatgaga ctctcactag agattagatg gagaaagggc 1800 tccagcaggc acggagctggaagctttgtc tgtgagacag ctccgcggga gcactcatcc 1860 cccagggctc tctgtctccctctgagaggc tctggcccca tntaaccacc agaatgggag 1920 aagaagtgct tccccgtgggattagggcac atctgtcccg caggcccacc tgcctgccag 1980 tccctcccag gattcctgcctggccacccc acaggagtgt gtacacagtg cagcctcagc 2040 tgctcagcat gggtgctttgctccacttga gtgcattccg gcagcgtggg agctgtttga 2100 atcccccagt gcacacagatcccaacccca agggtccagg ggagggagct gtgagcagat 2160 ccggacgtcc cagggctgtggctccggagt gcggaactgg gcccagtgct tcagcagaag 2220 aggagcccat actctcagaaaactctcaga gaggggtgag tngnacaggt tcctgggctg 2280 gtgtggaacc tangcgtgcctncctncaca gagctggtcc agtaagtgtg gggcctgtct 2340 ccctgctgga cctctgcctgaaggagccca acgacctgga acacctaaca acaacagaaa 2400 gtcncggcca cagtgccagtgatcaggggt ccctcccctc aagaccgagg aggagacctg 2460 gtgaggggtc acccctctcccccttgcacc acagagcacg gcttcaaagg cccggataca 2520 caaaggagcc gggtggcagaatattagtct agctatctcc cattgctctc acgcgccatc 2580 tactggattt catcccaaactacaacacga aaaactgcta attttcctgc ctgccaggcc 2640 gaggactgga attcaacagactgtttagag cctttgccct ctgaaaactt ccagaaatga 2700 agccaactga ctatattcagtttacaccag agttaaagga acgccaaccc tcccagatga 2760 gaaagaatca gtgcaagaactgtagcaatt taaaaaacca gagcgtcccc ttacctccaa 2820 atgagcccac tagctccacagcaattgttc ttaaccaatc tgaaatgatg agcatggaat 2880 tcagaatctg aatggcaatgaagcttatag atatccagga gaaagttgaa atgcaatcca 2940 aggaaaccaa gcaatccagtgaaatggttt aagagctgaa agataaaata ncaattttac 3000 aaaagaccca aactgagcttattgagttca aaaaagaatt tcataataca atcagaagta 3060 ttaatagcag aataggccaagctgaggaaa gaatctcaga gcttgacccc tggttctttg 3120 aatcaactta gacaaaaataaagaaaaaag agttttaaga aatgaacaca atctcccaga 3180 aatatgagat tatgtwaagagacaaaatct atgactcatt gccatccctg agagagaagg 3240 agagagaata agcaacttggaaaatatatt tggggacata gcccacaaaa atttccctaa 3300 tctctctaga gaggttgacatgtaaattca agaaatacag aagaccttgg ccagataata 3360 tacaagatga ccatccccaaggcacatagt catcagattc accatggtca atgcaaaaga 3420 aaaaaatctt aaagacagctagggagaagg gtcaagtcac atgcagaagg actctcatta 3480 ggctggcagt ggacctctcagcagaaacct gacaagccag aagagatgga gggagagggg 3540 tctatttttg tcatccttaaagaaaaaaaa ttccaaccaa gagtctcata cactgccaaa 3600 ctaagcttcc taagtgaaggagaaataaaa accttctcag acaagcaaat gctgaaggaa 3660 ttcaactaga ccagcctaacaagaggtcct aagggagtgc tgaatatgga ctcaaaagaa 3720 taacacctgc taccacaaacactcacttaa gcacacagcc caacgacact ataggcaatt 3780 acacagtaag tctacataacaacacaatga caggatcaac atctcacaca tcaatactaa 3840 ccccgagtgt aaaggggctaaatgccccac ttaaaagaca tagagtgtca agcttgataa 3900 aaagacaaga tccaatcatccactattttc aagagctcta tgttatgtgt aatgacaccc 3960 acagactcaa agacttggagaaagatttat catgcaaaat cagaaaacaa aaaagagcag 4020 gagtcactag ttttatatcagacaaaacag actttaaacc cttaataatt aagaaagaca 4080 aagaagggta tttcctggaccacagaaggc ttattggaaa aaaggacata atgacaaagg 4140 gtacaatcca acaagaagttttaactattc taaatatata cacacccaac attggagcac 4200 ccagatttat aaaacaagtacttctcgatc tacaagaaga cttagacagc cacacaataa 4260 tagtgggaga ctttcacatcctacttacag atcattgaga cagaaaacta ataaaagaac 4320 tctggactta aacttgttacttgaccaatt ggacctaata gatatccaca gaaaacttca 4380 cccaacaaag acagaatatacattcttctt atctgcacat ggaacacatt ccaagatcaa 4440 tcacatgcta ggtaagaaagcaagtctcaa taaattaaaa aaaattgaaa tcatacgaac 4500 cttaatatca gaccacaatgtaattaaaaa taaatcaata tcaagaagat ctcatacata 4560 aatacatgaa aattaaacaacttactcctg aataactctt gtgtgaacat caaaattcag 4620 gaagaaataa aaaattatttgaaatt 4646 68 2484 DNA Homo sapiens any n or Xaa = unknown 68tcttgaccgg cacacacagc tcgcttcttc actttctttt ccatccactg ccggacccaa 60gccagccttc cagggagcag ccatgcctta cctctaccgg gccccagggc ctcaggcaca 120cccggttccc aaggacgccc ggatcaccca ctcctcaggc cagagctttg agcaaatgag 180gcaggagtgc ctgcagagag gcaccctgtt tgaggatgca gacttcccag ccagcaattc 240ctccctgttc tacagtgaga ggccgcagat cccctttgtg tggaaacgac caggggaaat 300cgtgaaaaac ccagaattca ttcttggagg ggccaccagg actgatatct gccagggaga 360gctgggagac tgctggctat tagccgccat cgcctccctt acgcttaatc aaaaagcact 420ggccagagtc atcccccagg accaaagctt tggccctggt tatgccggga tattccattt 480ccagttctgg cagcacagtg agtggctgga cgtggtgatc gatgaccgcc tgcccacctt 540cagggaccgc ttggttttcc tccactctgc cgaccacaac gagttctgga gcgccttgct 600ggaaaaagcc tacgccaagc taaatgggag ctatgaagct ctgaagggag gcagcgccat 660cgaggccatg gaagacttca ctgggggtgt ggcagagacc ttccaaacta aagaggcccc 720cgagaacttc tatgagattc tagagaaggc tttgaagaga ggctccctgc tgggctgctt 780cattgatacc agaagtgctg cagaatctga ggcccggacg ccgtttggtc ttattaaggg 840tcatgcctac agtgtaacgg gaattgacca ggtaagcttc cgaggccaga gaatcgagct 900catccgaatc cggaaccctt ggggccaggt tgagtggaac gggtcgtgga gcgacaggat 960ggcatttaag gacttcaagg cccactttga taaagtggag atctgcaacc tcactcccga 1020tgccctggag gaagacgcga tccacaaatg ggaggtgacg gtccatcagg gaagctgggt 1080tcgcggctcc acggctgggg gctgccgcaa tttcctggat accttttgga ccaatccaca 1140aataaaattg tctctgactg agaaagatga ggggcaggag gagtgtagtt tccttgtagc 1200cctgatgcag aaagatagaa ggaaactcaa gagatttggt gccaatgtgc tgacaatcgg 1260ctatgccatt tatgagtgcc ctgacaaaga cgaacacctg aacaaagact tcttcagata 1320ccacgcttct cgggccagaa gcaagacgtt catcaacctg agagaagtct ccgaccggtt 1380caagctgccc cctggggagt acatcctgat tcccagcact tttgagcccc accaggaagc 1440tgatttctgt ctgagaatct tttcagagaa aaaagccatt acccgggata tggatggaaa 1500tgtagacatt gaccttcctg agcctccaaa gccaactcca cctgaccagg agacagagga 1560ggagcagcgg tttcgggctc tgtttgaaca agtcgctggt gaggacatgg aggtgacagc 1620agaggaactt gagtatgttt taaatgctgt gctgcaaaag aaaaaggaca tcaaattcaa 1680gaagctaagc ctgatctcct gtaaaaacat catttccctg atggacacca gcggcaatgg 1740gaagctggag tttgatgaat tcaaagtgtt ctgggacaag ctgaagcagt ggattaacct 1800tttccttcgg tttgatgctg acaagtccgg caccatgtct acctatgaac tacggactgc 1860actgaaagct gcaggctttc agctgagcag ccacctcctg cagctgattg tgctcaggta 1920tgcggatgag gagctccagc tggacttcga tgacttcctc aactgcctgg tccggctgga 1980gaatgcgagc cgggtgttcc aggctctcag tacaaagaac aaggagttca ttcatctcaa 2040tataaatgag ttcatccatt tgacaatgaa catctgaggc tgccttgtag agatgcagcc 2100tgcccagctg aatcttggct tctggacctt gaccttcaga acttctcttg gtgtggaacc 2160attacgccca gggttcactc ccctctcatc gtccggcctt ctcccttcat cttgatctgg 2220gaagaatgaa atgaactcag ctacactctc tgattttgtg ctactccttt gtaaagtcac 2280tgccttaagg gggctgatgg cgccacctgt gccttacatc caggttcagg catcactagc 2340tttcccacac tctactttcc ttatttcctt ccattaagaa ttactcagag ttctaacgca 2400cagaatcctg acttccatgt agctccagtc attgtgatca gacatccttt ataaaacatg 2460tttttataaa tgtgtatgtg gaat 2484 69 199 PRT Homo sapiens any n or Xaa =unknown 69 Met Leu Ala Tyr Ser Ser Val His Cys Phe Arg Glu Asp Lys MetLys 1 5 10 15 Phe Thr Ile Val Phe Ala Gly Leu Leu Gly Val Phe Leu AlaPro Ala 20 25 30 Leu Ala Asn Tyr Asn Ile Asn Val Asn Asp Asp Asn Asn AsnAla Gly 35 40 45 Ser Gly Gln Gln Ser Val Ser Val Asn Asn Glu His Asn ValAla Asn 50 55 60 Val Asp Asn Asn Asn Gly Trp Asp Ser Trp Asn Ser Ile TrpAsp Tyr 65 70 75 80 Gly Asn Gly Phe Ala Ala Thr Arg Leu Phe Gln Lys LysThr Cys Ile 85 90 95 Val His Lys Met Asn Lys Glu Val Met Pro Ser Ile GlnSer Leu Asp 100 105 110 Ala Leu Val Lys Glu Lys Lys Leu Gln Gly Lys GlyPro Gly Gly Pro 115 120 125 Pro Pro Lys Gly Leu Met Tyr Ser Val Asn ProAsn Lys Val Asp Asp 130 135 140 Leu Ser Lys Phe Gly Lys Asn Ile Ala AsnMet Cys Arg Gly Ile Pro 145 150 155 160 Thr Tyr Met Ala Glu Glu Met GlnGlu Ala Ser Leu Phe Phe Tyr Ser 165 170 175 Gly Thr Cys Tyr Thr Thr SerVal Leu Trp Ile Val Asp Ile Ser Phe 180 185 190 Cys Gly Asp Thr Val GluAsn 195 70 664 PRT Homo sapiens any n or Xaa = unknown 70 Met Pro TyrLeu Tyr Arg Ala Pro Gly Pro Gln Ala His Pro Val Pro 1 5 10 15 Lys AspAla Arg Ile Thr His Ser Ser Gly Gln Ser Phe Glu Gln Met 20 25 30 Arg GlnGlu Cys Leu Gln Arg Gly Thr Leu Phe Glu Asp Ala Asp Phe 35 40 45 Pro AlaSer Asn Ser Ser Leu Phe Tyr Ser Glu Arg Pro Gln Ile Pro 50 55 60 Phe ValTrp Lys Arg Pro Gly Glu Ile Val Lys Asn Pro Glu Phe Ile 65 70 75 80 LeuGly Gly Ala Thr Arg Thr Asp Ile Cys Gln Gly Glu Leu Gly Asp 85 90 95 CysTrp Leu Leu Ala Ala Ile Ala Ser Leu Thr Leu Asn Gln Lys Ala 100 105 110Leu Ala Arg Val Ile Pro Gln Asp Gln Ser Phe Gly Pro Gly Tyr Ala 115 120125 Gly Ile Phe His Phe Gln Phe Trp Gln His Ser Glu Trp Leu Asp Val 130135 140 Val Ile Asp Asp Arg Leu Pro Thr Phe Arg Asp Arg Leu Val Phe Leu145 150 155 160 His Ser Ala Asp His Asn Glu Phe Trp Ser Ala Leu Leu GluLys Ala 165 170 175 Tyr Ala Lys Leu Asn Gly Ser Tyr Glu Ala Leu Lys GlyGly Ser Ala 180 185 190 Ile Glu Ala Met Glu Asp Phe Thr Gly Gly Val AlaGlu Thr Phe Gln 195 200 205 Thr Lys Glu Ala Pro Glu Asn Phe Tyr Glu IleLeu Glu Lys Ala Leu 210 215 220 Lys Arg Gly Ser Leu Leu Gly Cys Phe IleAsp Thr Arg Ser Ala Ala 225 230 235 240 Glu Ser Glu Ala Arg Thr Pro PheGly Leu Ile Lys Gly His Ala Tyr 245 250 255 Ser Val Thr Gly Ile Asp GlnVal Ser Phe Arg Gly Gln Arg Ile Glu 260 265 270 Leu Ile Arg Ile Arg AsnPro Trp Gly Gln Val Glu Trp Asn Gly Ser 275 280 285 Trp Ser Asp Arg MetAla Phe Lys Asp Phe Lys Ala His Phe Asp Lys 290 295 300 Val Glu Ile CysAsn Leu Thr Pro Asp Ala Leu Glu Glu Asp Ala Ile 305 310 315 320 His LysTrp Glu Val Thr Val His Gln Gly Ser Trp Val Arg Gly Ser 325 330 335 ThrAla Gly Gly Cys Arg Asn Phe Leu Asp Thr Phe Trp Thr Asn Pro 340 345 350Gln Ile Lys Leu Ser Leu Thr Glu Lys Asp Glu Gly Gln Glu Glu Cys 355 360365 Ser Phe Leu Val Ala Leu Met Gln Lys Asp Arg Arg Lys Leu Lys Arg 370375 380 Phe Gly Ala Asn Val Leu Thr Ile Gly Tyr Ala Ile Tyr Glu Cys Pro385 390 395 400 Asp Lys Asp Glu His Leu Asn Lys Asp Phe Phe Arg Tyr HisAla Ser 405 410 415 Arg Ala Arg Ser Lys Thr Phe Ile Asn Leu Arg Glu ValSer Asp Arg 420 425 430 Phe Lys Leu Pro Pro Gly Glu Tyr Ile Leu Ile ProSer Thr Phe Glu 435 440 445 Pro His Gln Glu Ala Asp Phe Cys Leu Arg IlePhe Ser Glu Lys Lys 450 455 460 Ala Ile Thr Arg Asp Met Asp Gly Asn ValAsp Ile Asp Leu Pro Glu 465 470 475 480 Pro Pro Lys Pro Thr Pro Pro AspGln Glu Thr Glu Glu Glu Gln Arg 485 490 495 Phe Arg Ala Leu Phe Glu GlnVal Ala Gly Glu Asp Met Glu Val Thr 500 505 510 Ala Glu Glu Leu Glu TyrVal Leu Asn Ala Val Leu Gln Lys Lys Lys 515 520 525 Asp Ile Lys Phe LysLys Leu Ser Leu Ile Ser Cys Lys Asn Ile Ile 530 535 540 Ser Leu Met AspThr Ser Gly Asn Gly Lys Leu Glu Phe Asp Glu Phe 545 550 555 560 Lys ValPhe Trp Asp Lys Leu Lys Gln Trp Ile Asn Leu Phe Leu Arg 565 570 575 PheAsp Ala Asp Lys Ser Gly Thr Met Ser Thr Tyr Glu Leu Arg Thr 580 585 590Ala Leu Lys Ala Ala Gly Phe Gln Leu Ser Ser His Leu Leu Gln Leu 595 600605 Ile Val Leu Arg Tyr Ala Asp Glu Glu Leu Gln Leu Asp Phe Asp Asp 610615 620 Phe Leu Asn Cys Leu Val Arg Leu Glu Asn Ala Ser Arg Val Phe Gln625 630 635 640 Ala Leu Ser Thr Lys Asn Lys Glu Phe Ile His Leu Asn IleAsn Glu 645 650 655 Phe Ile His Leu Thr Met Asn Ile 660

What is claimed is:
 1. A method for detecting a cancer cell in aresected specimen, characterized by determining a change in anexpression level of at least one of cancer-associated genes selectedfrom genes of which cDNA is a DNA comprising a nucleotide sequence asshown in any one of SEQ ID NOs: 1 to 16 and 66 to 68 in SequenceListing, or a DNA capable of hybridizing with a nucleic acid consistingof a nucleotide sequence as shown in any one of SEQ ID NOs: 1 to 16 and66 to 68 in Sequence Listing under stringent conditions.
 2. Thedetection method according to claim 1, characterized in that the changein an expression level of a cancer-associated gene is determined by thechange in an expression level of mRNA corresponding to said gene.
 3. Thedetection method according to claim 2, characterized in that the changein an expression level of mRNA is detected by utilizing a nucleic acidamplification method based on said mRNA or a partial portion thereof. 4.The detection method according to claim 3, characterized in that saidnucleic acid amplification is polymerase chain reaction.
 5. Thedetection method according to claim 2, characterized in that the changein an expression level of mRNA is detected by Northern hybridizationmethod.
 6. The detection method according to claim 2, characterized inthat the change in an expression level of mRNA is detected by RNaseprotection assay.
 7. The detection method according to claim 1,characterized in that the change in an expression level of acancer-associated gene is determined by a change in an expression levelof a protein encoded by said gene.
 8. The detection method according toclaim 7, characterized in that the change in expression of the proteinis detected by utilizing an antibody capable of recognizing saidprotein.
 9. A kit for detecting cancer by the method of claim 3, whereinthe kit comprises primers for amplifying mRNA of which change in anexpression level is to be determined or a partial portion thereof.
 10. Akit for detecting a cancer cell by the method of claim 8, wherein thekit comprises an antibody recognizing a protein of which change in anexpression level is to be determined.
 11. A method for controllingproliferation of a cancer cell using a substance specifically binding toa gene or an expression product of said gene, characterized in that cDNAof the gene is at least one of DNAs selected from a DNA comprising anucleotide sequence as shown in any one of SEQ ID NOs: 1 to 16 and 66 to68 in Sequence Listing, or a DNA capable of hybridizing with a nucleicacid consisting of any one of these nucleotide sequences under stringentconditions, wherein the DNA is usable for detection of a cancer cell bya change in an expression level thereof.
 12. A peptide usable fordetection of a cancer cell, characterized in that the peptide is shownin an amino acid sequence comprising an entire portion of any one ofamino acid sequences as shown in SEQ ID NOs: 17 to 19, 69 and 70 inSequence Listing or a partial portion thereof.
 13. A peptide usable fordetection of a cancer cell, characterized in that the peptide has anamino acid sequence comprising an amino acid sequence resulting from atleast one of deletion, substitution or addition of one or more aminoacid residues in an amino acid sequences as shown in any one of SEQ IDNOs: 17 to 19, 69 and 70 in Sequence Listing.
 14. An antibody usable fordetection of a cancer cell, wherein the antibody recognizes the peptideof claim 12 or
 13. 15. A kit for detecting a gastric cancer cell,wherein the kit comprises primer pairs which are capable of amplifying amRNA for a cancer-associated gene to be detected of which change in anexpression level is to be determined, wherein said cancer-associatedgene consists of the nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1 and 66, and wherein the primer pairs consistof at least two primers, each comprising 10 to 30 nucleotides.